Characterization of the self association of Avian sarcoma virus integrase by analytical ultracentrifugation.

Retroviral integration protein (IN) has been shown to be both necessary and sufficient for the integration of reverse-transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-assembly of IN is essential for proper function. Analytical ultracentrifugation was used to determine the stoichiometry and free energy of self-association of a full-length IN in various solvents at 23.3 degrees C. Below 8% glycerol, an association stoichiometry of monomer-dimer-tetramer is observed. At salt concentrations above 500 mM, dimer is the dominant species over a wide range of protein concentrations. However, as physiological salt concentrations are approached, tetramer formation is favored. The addition of glycerol to 500 mM NaCl, 20 mM Tris (pH 8.4), 2 mM beta-mercaptoethanol significantly enhances dimer formation with little effect on tetramer formation. Furthermore, as electrostatic shielding is increased by increasing the ionic strength or decreasing the cation size, dimer formation is strengthened while tetramer formation is weakened. Taken together, the data support a model in which dimer formation includes favorable buried surface interactions which are opposed by charge-charge repulsion, while favorable electrostatic interactions contribute significantly to tetramer formation.     

Effects of [K(+)](o) on electrical restitution and activation dynamics during ventricular fibrillation

To test whether hyperkalemia suppresses ventricular fibrillation (VF) by reducing the slope of the action potential duration (APD) restitution relation, we determined the effects of the extracellular K(+) concentration ([K(+)](o)) ([KCl] = 2.7-12 mM) on the restitution of APD and maximum upstroke velocity (V(max)) the magnitude of APD alternans and spatiotemporal organization during VF in isolated canine ventricle. As [KCl] was increased incrementally from 2.7 to 12 mM, V(max) was reduced progressively. Increasing [KCl] from 2.7 to 10 mM decreased the slope of the APD restitution relation at long, but not short, diastolic intervals (DI), decreased the range of DI over which the slope was >/=1, and reduced the maximum amplitude of APD alternans. At [KCl] = 12 mM, the range of DI over which the APD restitution slope was >/=1 increased, and the maximum amplitude of APD alternans increased. For [KCl] = 4-8 mM, the persistence of APD alternans at short DI was associated with maintenance of VF. For [KCl] = 10-12 mM, the spontaneous frequency during VF was reduced, and activation occurred predominantly at longer DI. The lack of APD alternans at longer DI was associated with conversion of VF to a periodic rhythm. These results provide additional evidence for the importance of APD restitution kinetics in the development of VF.     

Analysis of the self-association of human red cell spectrin

The self-association equilibrium of spectrin has been studied by separating the molecular species present in the cooled reaction mixture by gel electrophoresis. The association constant for formation of the hexamer from dimer and tetramer is lower by an order of magnitude than that for the association of two dimers. The association constant for the formation of the octamer from the hexamer is appreciably larger, and the value appears to reach a constant level for higher oligomers. These observations are explained in terms of conformational strain due to formation of cyclic structures, the distortion being greatest on passing from the tetramer to the hexamer. The association for a single-site interaction between the dimer and a univalent fragment has also been analyzed. The results show that the free energy generated by a single-point interaction is much greater than that obtained by averaging over all pairwise interactions within the oligomers, correcting for the effect of cratic entropy. The results are related to the association state of the spectrin prevailing in the cell. Phosphorylation at the physiological sites in the dimer does not appreciably change the thermodynamics of self-association, at least up to the hexamer.     

Activation of the human glucocorticoid receptor: evidence for a two-step model

The relationship between glucocorticoid receptor subunit dissociation and activation was investigated by DEAE-cellulose and DNA-cellulose chromatography of monomeric and multimeric [3H]triamcinolone acetonide ([3H]TA)-labeled IM-9 cell glucocorticoid receptors. Multimeric (7-8 nm) and monomeric (5-6 nm) complexes were isolated by Sephacryl S-300 chromatography. Multimeric complexes did not bind to DNA-cellulose and eluted from DEAE-cellulose at a salt concentration (0.2 M KCl) characteristic of unactivated steroid-receptor complexes. Monomeric [3H]TA-receptor complexes eluted from DEAE-cellulose at a salt concentration (20 mM KCl) characteristic of activated steroid-receptor complexes. However, only half of these complexes bound to DNA-cellulose. This proportion could not be increased by heat treatment, addition of bovine serum albumin, or incubation with RNase A. Incubation of monomeric complexes with heat inactivated cytosol resulted in a 2-fold increase in DNA-cellulose binding. Unlike receptor dissociation, this increase was not inhibited by the presence of sodium molybdate. Fractionation of heat inactivated cytosol by Sephadex G-25 chromatography demonstrated that the activity responsible for the increased DNA binding of monomeric [3H]TA-receptor complexes was macromolecular. These results are consistent with a two-step model for glucocorticoid receptor activation, in which subunit dissociation is a necessary but insufficient condition for complete activation. They also indicate that conversion of the steroid-receptor complex to the low-salt eluting form is a reflection of receptor dissociation but not necessarily acquisition of DNA-binding activity.     

Histone subunit interactions as investigated by high pressure

High-pressure fluorescence polarization was used to investigate subunit interactions of the histone H2A-H2B dimer and the H3/H4 tetramer isolated from calf thymus (CT) and chicken erythrocyte (CE) chromatin. The proteins were individually labeled with the fluorescent probe 5-(dimethylamino)-naphthalene-1-sulfonate (dansyl or DNS), and the fluorescence polarization was measured as a function of pressure. The long fluorescence lifetime of the probe allows for the observation of global rotations of the protein, the rate of which is dependent upon the aggregation state. From the pressure dependence of the dansyl polarization, the Kd of H2A-H2B dissociation of the CE dimer was found to be approximately 1 X 10(-7) M at 2.0 M NaCl. Lowering the salt concentration to 200 mM slightly stabilized the protein to 6 X 10(-8) M. Our data indicate a small negative volume change for the dissociation of the core particle octamer. The (H3)2(H4)2 tetramer, as was shown in the previous paper (Royer et al., 1989), also formed predominantly dimers of tetramers at higher protein or salt concentrations. In the study presented here, we found the dissociation constant for the H3/H4 octamer to dimer transition to be 1 X 10(-21) M3 (C1/2 = 4 X 10(-8) M) at 2 M NaCl for the CT preparation. Decreasing the salt concentration to 200 mM reduced the stability of the CT H3/H4 octamer to 9 X 10(-21) M3 (C1/2 = 8 X 10(-8) M). The dimer of the CE tetramer also dissociated upon application of pressure in 2 M salt.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stoichiometric association of cap-binding protein I with translated polysomal globin mRNP.

The protein composition of a 12S polysomal globin messenger ribonucleoprotein (pmRNP) from rabbit reticulocytes was examined. The pmRNP was released from purified polysomes by puromycin treatment under run-off conditions of protein synthesis. The protein pattern of this pmRNP depends on the potassium ion concentration used during the run-off and the subsequent isolation. Several proteins show a salt-dependent association with the pmRNP while a few are constituents of the pmRNP at all salt concentrations tested. By cross-linking the pmRNP-derived proteins to [3H]methyl-labelled oxidized vesicular stomatitis virus (VSV) mRNA and by immunoblotting against anti-cap-binding protein (CBP I) antibodies, it is demonstrated that the association of the CBP I with the pmRNP depends on the ionic strength. At 65 mM KCl, CBP I shows low affinity for the pmRNP; at 140 mM KCl, the affinity of CBP I for the pmRNP is greatly enhanced. At this ionic strength, equimolar amounts of CBP I and mRNA are found in the pmRNP. At 500 mM KCl, the pmRNP is completely devoid of CBP I. In the non-translated free cytoplasmic mRNP (cmRNP) no CBP can be detected by either the cross-link or the immunoblot technique.     

Regulation of activity of the yeast TATA-binding protein through intra-molecular interactions

Dimerization is proposed to be a regulatory mechanism for TATA-binding protein (TBP) activity bothin vitro andin vivo. The reversible dimer-monomer transition of TBP is influenced by the buffer conditionsin vitro. Usingin vitro chemical cross-linking, we found yeast TBP (yTBP) to be largely monomeric in the presence of the divalent cation Mg²⁺, even at high salt concentrations. Apparent molecular mass of yTBP at high salt with Mg²⁺, run through a gel filtration column, was close to that of monomeric yTBP. Lowering the monovalent ionic concentration in the absence of Mg²⁺, resulted in dimerization of TBP. Effect of Mg²⁺ was seen at two different levels: at higher TBP concentrations, it suppressed the TBP dimerization and at lower TBP levels, it helped keep TBP monomers in active conformation (competent for binding TATA box), resulting in enhanced TBP-TATA complex formation in the presence of increasing Mg²⁺. At both the levels, activity of the full-length TBP in the presence of Mg²⁺ was like that reported for the truncated C-terminal domain of TBP from which the N-terminus is removed. Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar mechanism.     

Human homologue of the Drosophila discs large tumor suppressor protein forms an oligomer in solution. Identification of the self-association site

The human homologue of the Drosophila discs large tumor suppressor protein (hDlg), a member of the membrane-associated guanylate kinase (MAGUK) superfamily, interacts with K(+) channels, N-methyl-d-aspartate receptors, calcium ATPase, adenomatous polyposis coli, and PTEN tumor suppressor proteins, and several viral oncoproteins through its PDZ domains. MAGUKs play pivotal roles in the clustering and aggregation of receptors, ion channels, and cell adhesion molecules at the synapses. To investigate the physiological basis of hDlg interactions, we examined the self-association state of full-length hDlg as well as defined segments of hDlg expressed as recombinant proteins in bacteria and insect Sf9 cells. Gel permeation chromatography of full-length hDlg revealed that the purified protein migrates as a large particle of size >440 kDa. Similar measurements of defined domains of hDlg indicated that the anomalous mobility of hDlg originated from its amino-terminal domain. Ultrastructural analysis of hDlg by low angle rotary shadow electron microscopy revealed that the full-length hDlg protein as well as its amino-terminal domain exhibits a highly flexible irregular shape. Further evaluation of the self-association state of hDlg using sedimentation equilibrium centrifugation, matrix-assisted laser desorption/ionization mass spectrometry, and chemical cross-linking techniques confirmed that the oligomerization site of hDlg is contained within its amino-terminal domain. This unique amino-terminal domain mediates multimerization of hDlg into dimeric and tetrameric species in solution. Sedimentation velocity experiments demonstrated that the oligomerization domain exists as an elongated tetramer in solution. In vitro mutagenesis was used to demonstrate that a single cysteine residue present in the oligomerization domain of hDlg is not required for its self-association. Understanding the oligomerization status of hDlg may help to explicate the mechanism of hDlg association with multimeric K(+) channels and dimeric adenomatous polyposis coli tumor suppressor protein. Our findings, therefore, begin to rationalize the role of hDlg in the clustering of membrane channels and formation of multiprotein complexes necessary for signaling and cell proliferation pathways.     

A model for the solution structure of the rod arrestin tetramer

Visual rod arrestin has the ability to self-associate at physiological concentrations. We previously demonstrated that only monomeric arrestin can bind the receptor and that the arrestin tetramer in solution differs from that in the crystal. We employed the Rosetta docking software to generate molecular models of the physiologically relevant solution tetramer based on the monomeric arrestin crystal structure. The resulting models were filtered using the Rosetta energy function, experimental intersubunit distances measured with DEER spectroscopy, and intersubunit contact sites identified by mutagenesis and site-directed spin labeling. This resulted in a unique model for subsequent evaluation. The validity of the model is strongly supported by model-directed crosslinking and targeted mutagenesis that yields arrestin variants deficient in self-association. The structure of the solution tetramer explains its inability to bind rhodopsin and paves the way for experimental studies of the physiological role of rod arrestin self-association.     

Analysis of the dynamic equilibrium of histone oligomers in a solution. The nature of forces stabilizing the (H2A-H2B-H3-H4)2 octamer structure

Dynamic equilibrium analysis of the (H2A-H2B-H3-H4)2 histone octamer with lower oligomers was performed in 2 M NaCl. Calculated data on the relative content of histone oligomers upon changing protein concentration in solution are given. The red shift of lambda max for histone tyrosine fluorescence spectra is shown to be due to hydrogen bond formation by tyrosyl OH-groups. Analysis of free energy changes of histone oligomers upon association (delta G = -17,37 +/- 0,14 kcal/mole) as well as the effect of urea on histone octamer dissociation made it possible to conclude that virtually all tyrosyls in octamer form hydrogen bonds. Intermolecular hydrogen bonds formed by tyrosyls contribute substantially to octamer stabilization. The (H2A-H2B) dimer positive cooperativity in association with the (H3-H4)2 tetramer was found. This cooperativity is caused by interaction between association sites with a two order increase in an apparent constant of dimers with tetramer association. The histone octamer was determined to be of asymmetric structure due to unequivolency of the two binding sites for the (H2A-H2B) dimers.     

The cell surface glycoprotein of Halobacterium halobium. Physico-chemical characterization in the absence and presence of salt

The cell surface glycoprotein of Halobacterium halobium is soluble in dilute buffer at neutral pH. At low counterion concentrations, the protein is monomeric (Ms,D = 209 kDa) and exhibits the characteristics of a highly charged polyelectrolyte. Evidence obtained from intrinsic fluorescence and far-UV circular dichroism shows that the monomer at low salt loses both its native conformation and its inherent tendency to form high molecular mass assemblies. In 4M NaCl, 25 mM KCl, and in the presence of divalent ions (greater than or equal to 50mM Mg2+ or Ca2+), association to well-defined assemblies of up to approximately 4 X 10(6) Da occurs. At low Mg2+ concentration and in the presence of Ba2+, a wide size-distribution of aggregates is observed. The assembly pattern of the protein may be correlated with salt-dependent alterations in the morphology of the bacterium.     

Influence of an anion-binding site in the stabilization of halophilic malate dehydrogenase from Haloarcula marismortui

Halophilic proteins have evolved to be soluble, stable and active in high salt concentration. Crystallographic studies have shown that surface enrichment by acidic amino acids is a common structural feature of halophilic proteins. In addition, ion-binding sites have also been observed in most of the cases. The role of chloride-binding sites in halophilic adaptation was addressed in a site-directed mutagenesis study of tetrameric malate dehydrogenase from Haloarcula marismortui. The mutation of K 205, which is involved in an inter-subunit chloride-binding site, drastically modified the enzyme stability in the presence of KCl, but not in the presence of KF. The oligomeric state of the [K205A] mutant changes with the nature of the anion. At high salt concentration, the [K205A] mutant is a dimer when the anion is a chloride ion, whereas it is a tetramer when the fluoride ion is used. The results highlight the role of anion-binding sites in protein adaptation to high salt conditions.     

The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.     

Hidden self-association of proteins

Sedimentation equilibrium measurements were carried out on solutions of bovine serum albumin, aldolase, and ovalbumin in phosphate-buffered saline, pH 7.2, at 10 degrees C. The data obtained for each protein were analyzed to yield the dependence of apparent weight-average molecular weight upon protein concentration, over a concentration range of ca 1-200 g/L. Using the approximate theory of Chatelier and Minton [1987) Biopolymers 26, 507-524), models are formulated for the dependence of apparent weight-average molecular weight upon concentration in non-ideal solutions containing proteins which may self-associate according to a monomer/n-mer or a monomer/dimer/tetramer scheme. The concentration dependence data for serum albumin may be accounted for, assuming either no self-association or weak monomer/dimer association. The data for aldolase may be accounted for assuming either weak monomer/dimer or weak monomer/trimer association. The data for ovalbumin may be accounted for assuming either weak monomer/trimer or weak monomer/dimer/tetramer association. The associations do not approach saturation at the highest concentrations studied, and the standard-state free energy changes accompanying self-association amount to less than 4 kcal/mol of intermolecular contacts, suggesting that non-specific clustering of protein molecules at high concentration rather than the formation of specific complexes is being observed.