Influence of divalent cations in protein crystallization.

We have tested the effect of several cations in attempts to crystallize the ligand-bound forms of the leucine/isoleucine/valine-binding protein (LIVBP) (M(r) = 36,700) and leucine-specific binding protein (LBP) (M(r) = 37,000), which act as initial periplasmic receptors for the high-affinity osmotic-shock-sensitive active transport system in bacterial cells. Success was achieved with Cd2+ promoting the most dramatic improvement in crystal size, morphology, and diffraction quality. This comes about 15 years after the ligand-free proteins were crystallized. Nine other different divalent cations were tried as additives in the crystallization of LIVBP with polyethylene glycol 8000 as precipitant, and each showed different effects on the crystal quality and morphology. Cd2+ produced large hexagonal prism crystals of LIVBP, whereas a majority of the cations resulted in less desirable needle-shaped crystals. Zn2+ gave crystals that are long rods with hexagonal cross sections, a shape intermediate between the hexagonal prism and needle forms. The concentration of Cd2+ is critical. The best crystals of the LIVBP were obtained in the presence of 1 mM CdCl2, whereas those of LBP, with trigonal prism morphology, were obtained at a much higher concentration of 100 mM. Both crystals diffract to at least 1.7 A resolution using a conventional X-ray source.     

Adsorption of divalent cations on DNA

The distribution of divalent ions in semidilute solutions of high-molecular-mass DNA containing both sodium chloride and strontium chloride in near-physiological conditions is studied by small-angle x-ray scattering and by small-angle neutron scattering. Both small-angle neutron scattering and small-angle x-ray scattering reveal a continuous increase in the scattering intensity at low q with increasing divalent ion concentration, while at high q the scattering curves converge. The best fit to the data is found for a configuration in which DNA strands of cross-sectional radius 10 angstroms are surrounded by a counterion sheath of outer radius approximately 13.8 angstroms, independent of the strontium chloride concentration. When the strontium chloride is replaced by calcium chloride, similar results are obtained, but the thickness of the sheath increases when the divalent salt concentration decreases. These results correspond in both cases to partial localization of the counterions within a layer that is thinner than the effective Debye screening length.     

DNA denaturation in situ. Effect of divalent cations and alcohols

Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.     

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.     

Separation of recombinant antibodies from DNA using divalent cations

New downstream methods for the purification of antibodies are required to meet the demands of current and future antibody applications, e.g. for mass production as cancer therapeutic. The standard chromatographic methods suffer from high material costs and mass transfer limitations. In this study, we established and characterized a method for DNA precipitation for antibody purification using divalent cations, particularly CaCl₂, using four different antibodies. By implementing high‐throughput screening using a factorial design plan, we determined that CaCl₂ concentration and PO₄^3? concentration were significant factors, while temperature and pH were not significant. We detected DNA precipitation as well as host‐cell protein (HCP) reduction. Two‐dimensional difference gel electrophoresis (2D‐DIGE) revealed that improved HCP removal does not occur via an unspecific random mechanism such as the enclosure of proteins in the precipitate. CaCl₂ precipitation of DNA and HCP can be combined with nonchromatographic methods such as precipitation and protein A affinity chromatography. This additional purification method not only enhances DNA removal, but also the removal of HCP and antibody multimers, which will reduce immunogenicity and increase homogeneity of the resulting drug.     

Intracellular divalent cations and neuronal excitability.

Itracellular injections of Mg into cat spinal motoneurones have a depolarizing action, associated with a fall in input conductance, and depression of the postspike hyperpolarizing after-potential (a.h.p.) as well as its underlying conductance increase. There is also an increase in excitability, sometimes leading to outright discharge, and a change in the current-firing relation: the normal primary range is largely abolished and the firing appears to have the characteristics of the normal secondary range. Intracellular effects of Mg are thus mainly opposite to those of Ca, possibly owing to competition at sites where Ca activates K channels. Intracellular injections of Mn also tend to depress the a.h.p. but have relatively little effect on resting potential and conductance, or action potentials. Co also depresses the a.h.p. but has a more pronounced depolarizing action, and produces particularly strong depression of action potentials. By contrast intracellular Sr tends to raise the membrane conductance and has a mild hyperpolarizing effect. During the injection of Sr, a.h.p's are depressed but this is followed by a rebound of increased a.h.p. amplitude and conductance. Unlike the other divalent cations tested, Sr strongly depressed excitatory postsynaptic potentials. In most respects Sr appears to behave like Ca.     

Condensation of nonstochiometric DNA/polycation complexes by divalent cations

This study found that divalent cations induced the further condensation of partially condensed DNA within nonstochiometric polycation complexes. The addition of a few mmol of a divalent cation such as calcium reduced by half the inflection point at which DNA became fully condensed by poly-L-lysine (PLL) and a variety of other polycations. The effect on DNA condensation was initially observed using a new method, which is based on the concentration-dependent self-quenching of fluorescent moieties (e.g., rhodamine) covalently linked to the DNA backbone at relatively high densities. Additional analyses, which employed ultracentrifugation, dynamic light scattering, agarose gel electrophoresis, and atomic force microscopy, confirmed the effect of divalent cations. These results provide an additional accounting of the process by which divalent cations induce greater chromatin compaction that is based on the representation of chromatin fibers as a nonstoichiometric polyelectrolyte complex. They also offer a new approach to assemble nonviral vectors for gene therapy.     

Aggregation of nucleosomes by divalent cations.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.     

Role of divalent cations on DNA polymorphism under low ionic strength conditions.

We have examined the conformational properties of poly(dG-m5dC) under a variety of low salt conditions and sample preparations. Extensive dialysis against 0.5 mM Na-cacodylate resulted in a left-handed polynucleotide conformation as determined by circular dichroism, in agreement with recently reported results. Similarly, extensive dialysis against Tris-EGTA also led to a left-handed conformation. Dilution of these samples led to a transition to the right-handed conformation. More stringent treatments such as dialysis followed by passage over an ion exchange column also resulted in a right-handed conformation. When these various solutions were examined using atomic absorption spectroscopy, significant levels of Mg+2 were observed (greater than or equal to 190 per 1000 nucleotides) in all samples showing a left-handed form, while much lower levels (less than or equal to 45 per 1000 nucleotides) were found in the low salt samples displaying a right-handed conformation. Addition of MgCl2 to samples in which divalent cations had been almost completely removed led to the reformation of the left-handed form. These results indicate that the left-handed form seen under certain low salt conditions is due to the presence of Mg+2 ions that remain bound to the polynucleotide, even in the presence of EDTA.     

Influence of divalent cations upon complement-mediated enzyme release from human polymorphonuclear leukocytes.

The complement component, C5a provokes the selective release of granule-associated enzymes from the intact, viable cytochalasin B-treated human polymorphonuclear leukocytes (PMN) in the absence of phagocytosis or cellular adherence to surfaces. Consquently, in this experimental system the influence of divalent cations on these two processes can be disregarded and their effects on enzymes secretion can be studied directly. Cytochalasin B-treated PMN exposed to C5a in calcium and magnesium-free media consistently secreted significant amounts of the granule-associated enzymes, beta-glucuronidase and lysozyme. The basal secretory response was not diminished if cells were preincubated with 5.0 mM EDTA, nor was it influenced if 1.0 mm or 2.0 mM EDTA were present in the reaction mixtures. The addition of calcium (up to 1.5 to 2.0 mM) produced a concentration-dependent enhancement of beta-glucuronidase release, whereas increasing amounts of calcium (above 2.0 mM) inhibited secretion of this enzyme. Lysozyme release was similarly enhanced by the addition of calcium, but inhibition with high concentrations was not observed. Calcium per se, in the absence of C5a, provoked only the release of lysozyme from these cells. The effects of calcium upon enzyme release were not associated with alterations in the state of assembly of cytoplasmic microtubules. These findings provide another example of the role of calcium in "stimulus-secretion coupling" and provide evidence that exocytosis of various granules in human PMN is regulated by independent mechanisms involving calcium.     

Electric birefringence of native DNA in an alternating field

The relaxation of the birefringence of native DNA in solution was investigated in a pulsed sine-wave electric field. Relaxation times were calculated from the degree of damping of the birefringence signal and were studied as a function of the strength and frequency of the applied field, the molecular weight of the DNA, and the viscosity and ionic strength of the solvent. Relaxation times decrease with increasing field strength. For high-molecular weight DNA (>10⁶ daltons), the relaxation times decreased with frequency and increased less than linearly with viscosity. For low-molecular-weight DNA (<6 × 10⁵ daltons), the relaxation times were independent of frequency, increased linearly with viscosity, and varied with the 1.65 ± 0.1 power of the molecular weight. The average birefringence of high-molecular-weight DNA decreased with frequency in 0.001M Na₂ EDTA plus NaOH, pH 7.0, but is much less frequency-dependent if the EDTA concentration is reduced tenfold, while the average birefringence of sonicated DNA increases in both solvents with increasing frequency.     

Electric birefringence of myosin subfragments.

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.     

Differential stability of DNA crossovers in solution mediated by divalent cations

The assembly of DNA duplexes into higher-order structures plays a major role in many vital cellular functions such as recombination, chromatin packaging and gene regulation. However, little is currently known about the molecular structure and stability of direct DNA–DNA interactions that are required for such functions. In nature, DNA helices minimize electrostatic repulsion between double helices in several ways. Within crystals, B-DNA forms either right-handed crossovers by groove–backbone interaction or left-handed crossovers by groove–groove juxtaposition. We evaluated the stability of such crossovers at various ionic concentrations using large-scale atomistic molecular dynamics simulations. Our results show that right-handed DNA crossovers are thermodynamically stable in solution in the presence of divalent cations. Attractive forces at short-range stabilize such crossover structures with inter-axial separation of helices less than 20 Å. Right-handed crossovers, however, dissociate swiftly in the presence of monovalent ions only. Surprisingly, left-handed crossovers, assembled by sequence-independent juxtaposition of the helices, appear unstable even at the highest concentration of Mg²⁺studied here. Our study provides new molecular insights into chiral association of DNA duplexes and highlights the unique role divalent cations play in differential stabilization of crossover structures. These results may serve as a rational basis to understand the role DNA crossovers play in biological processes.     

Organization in the cell nucleus: divalent cations modulate the distribution of condensed and diffuse chromatin

The organization of rat liver nuclei in vitro depends on the ionic milieu. Turbidity measurements of nuclear suspensions in the presence of varying concentrations of divalent cations have been correlated with nuclear ultrastructure. The concentration of MgCl2 (2 mM) at which turbidity of nuclear suspensions is maximal and chromatin condensation appears most extensive is the same concentration that reportedly (Gottesfeld et al., 1974, Proc. Natl. Acad. Sci. U. S. A. 71:2193-2197) precipitates "inactive" chromatin. Thus, a mechanism is suggested by which chromatin activity and ultrastructural organization within the nucleus may be mediated. The nuclear organizational changes attendant upon the decrease in divalent cation concentration were not entirely reversible.     

The binding of divalent cations to myosin.

Centrifuge transport, equilibrium dialysis, and electron paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of 10(6) M-1 (Class I) and 10(3) M-1 (Class II), respectively. At 0.6 M KCl concentration, the affinity of Mn2+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca greater than Mg = Zn = Co greater than Sr greater than Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37 degrees partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PPi, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.     

Influence of divalent cations on the reconstituted ADP, ATP exchange

1. Divalent cations cause a decrease in the exchange activity of the reconstituted ADP,ATP translocator from beef heart mitochondria. This effect is due to complex formation with the adenine nucleotides. 2. It is confirmed that only the free nucleotides are transported. A possible competition of free adenine nucleotides and the Mg2+-complexes for the binding site at the carrier protein is excluded. 3. The stability constants (Kn) for the cation-nucleotide complexes are derived from these experiments. For Mg2+-ATP, Kn = 0.8 x 10(4) M-1 and for Mg2+-ADP, Kn = 0.8 x 10(3) M-1 is obtained. 4. The carrier system was reconstituted with the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine. Interaction of the divalent cations with these phospholipids seem not to be important for the exchange suppression.     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.