Quasielastic light scattering by biopolymers. V. Interparticle interactions between polynucleosomes

Quasielastic light scattering and electrophoretic light scattering experiments were performed on chicken erythrocyte polynucleosome solutions at various temperatures and ionic strengths. The apparent diffusion coefficient, D_app, was found to depend on the scattering vector K. In general, D_app can be described as a damped oscillatory function of K in the ionic strength range of 10 to 60 mM and over the temperature range of 10 to 40°C. Electrophoretic light scattering studies on total digest chromatin samples indicate the apparent charge on the polynucleosomes increases as the ionic strength is lowered from 10 to 1 mM. These data are interpreted in terms of fluctuations in the surface charge distribution of the polyion and subsequent inducement of an asymmetric distribution of small ions about the polyion. These fluctuation components lead to the formation of “clusters” of polyions.     

Assembly of a spherical plant virus.

The conditions previously reported as necessary for the reassembly of spherical viruses have been distinctly unphysiological and such reassembly cannot be related directly to the in vivo reaction. Mild conditions for the in vitro reassembly of cowpea chlorotic mottle virus (CCMV) from its isolated components have now been described (Adolph & Butler 1975) and the reassembled virus characterized. This reassembly involved the co-aggregation of the RNA and protein around neutrality and at ionic strength 0.2, giving yields of 70% encapsidation at pH 6.0. The reaction was independent of temperature over the range 5-25 degrees C and did not require the presence of Mg2+ ions. The reassembled virus shows a stability similar to that of native CCMV, with the same change in sedimentation coefficient around pH 6.5. The molecular mass and buoyant density in CsCl are also the same as those of native CCMV, while the electron microscope reveals a surface morphology on the reassembled particles like that on native CCMV. Analysis of the number-average, mass-average, and Z-average molecular masses of the purified protein at both pH 6.0 and pH 7.5 suggests that the active unit for reassembly is a dimer of the protein subunit.     

Kinetic study of the self-assembly of brome mosaic virus capsid

The protein of brome mosaic virus can self assemble in-vitro to form empty capsids. In the absence of RNA at pH=7 and 0.5 M KCl there is a dynamic equilibrium between monomers and oligomers. At pH=5 the protein assembles into empty capsids. The kinetics of this assembly, triggered by pH jump from neutral to acidic pH, was investigated by X-ray and light scattering.Cryoelectron microscopy observations suggested that reconstitution is achieved by progressive incorporation of small building units in a spherical shell. This hypothesis has been tested by the analysis of the scattering data in terms of four classes of incomplete capsids represented as spherical shells with holes of different sizes. The time dependence of the population of each class was determined by a least squares analysis of the experimental data. Although the basic polymerizing unit has not been uniquely characterized, the results are compatible with a dimer for this species. The characteristic times for capsid assembly are found to vary as the inverse of the square of the concentration.     

Dynamic light-scattering study of synthetic myosin filaments

Synthetic myosin filaments of rabbit were prepared. Electron microscopy showed that the number-average length (L_n = 470 nm) and sharpness in length distribution (L_w/L_n = 1.03₆) were independent of ionic strengths of 134, 74, and 44 mM, whereas the number ratio of M-filaments (about 15 nm in diameter at the bare zone) to m-filaments (about 10 nm) strongly depended on ionic strength (IS); the major filaments were M-filaments at IS = 134 mM, m-filaments at IS = 74 mM, and almost exclusively m-filaments at IS = 44 mM. Dynamic light scattering showed that the change in diameter with the change in ionic strength by 2-h dialysis was reversible. Combination of dynamic light scattering and sedimentation studies suggested a dynamic equilibrium between M- and m-filaments. Dynamic light-scattering spectra at IS = 134 and 74 mM could be analyzed by a theory for rigid rods, whereas those at IS = 44 mM only by introducing semiflexibility of filaments; m-filaments are more flexible at IS = 44 than at 74 mM.     

Conformation of LiDNA in solutions of LiCl

We have derived radii of gyratin, R_g, from the absolute intensity of the scattered light of mondisperse linear Col E₁ LiDNA in solution at various LiCl concentrations up to 5M. The second virial coefficients, A₂, decrease strongly with increasing LiCl concentration, and vanish between 3 and 5M LiCl. It was thus possible to calculate a limiting value at a high salt concentration of 28.5 nm for the persistence length, a₀, of LiDNA, without the necessity of applying excluded-volume corrections. The value obtained is in good agreement with the value previously obtained for NaDNA at high NaCl concentrations, and can be identified with the high salt limit of DNA flexibility, with long-range electrostatic interactions effectively screened. Sedimentation coefficients in the ultracentrifuge and apparent and translational diffusion coefficients (at finite and vanishing scattering vectors, respectively) from dynamic laser-light scattering have also been obtained up to 5M LiCl. From the sedimentation and apparent diffusion, D(90), (at 90° scattering angle only) above 5M, and up to 9M LiCl, it could be shown that the solutions are stable for reasonable periods of time, and the molecular parameters vary smoothly and moderately at high salt. Conformational transitions were not observed and precipitation occurs between 9 and 10M LiCl.     

Structure and assembly of turnip crinkle virus. II. Mechanism of reassembly in vitro

Dissociation of turnip crinkle virus (TCV) at elevated pH and ionic strength produces free dimers of the coat protein and a ribonucleoprotein complex that contains the viral RNA, six coat-protein subunits, and the minor protein species, p80 (a covalently linked coat-protein dimer). This "rp-complex" is stable for several days in high salt at pH 8.5. Reassembly of TCV can be accomplished under physiological conditions, using isolated coat protein and either rp-complex or protein-free RNA. If rp-complex is used in reassembly, the same subunits remain bound to RNA on subsequent dissociation; if free RNA is used, rp-complex is regenerated. In both cases, the assembly is selective for viral RNA in competition experiments with heterologous RNA. Electron microscopy shows that assembly proceeds by continuous growth of a shell from an initiating structure, rather than by formation of distinct intermediates. We suggest that rp-complex is the initiating structure, suggest a model based on the organization of the TCV particle, and propose a mechanism for TCV assembly.     

Synthesis and characterization of an ionizable polyhexapeptide collagen model

Poly(Lys(HBr)-Gly-Pro-Pro-Gly-Pro) has been synthesized and studied by circular dichroism (CD) spectroscopy. It is apparently the first polyhexapeptide collagen model reported with an ionizable side chain. The monomer (ε-(p-nitrobenzyloxycarbonyl)-Lys-Gly-Pro-Pro-Gly-Pro-p-nitrophenyl-ester) was prepared by a stepwise strategy employing active esters. Polymerization in N,N-dimethyl formamide, followed by removal of the Lys side chain protection with HBr/acetic acid, gave a polydisperse product. Fractionation was accomplished by gel filtration chromatography. The polydisperse material had a molecular weight (M_r = 5–17,000). High molecular weight fractions from triple helices under concentrated conditions at 2°C. The triple helical structure gives a CD pattern very similar to that of collagen and its triple helical analogs. However, unlike collagen, the polyhexapeptide undergoes spontaneous dissociation at temperatures substantially below the melting temperature from a triple helical form to single chains. This process is promoted at low concentrations, high temperature, neutral pH, and low molecular weight, and is apparently due, in large part, to unfavorable ionic side-chain interactions. In addition to this relatively slow “ionic” dissociation the triple helical polypeptide may be thermally dissociated in a manner similar to collagen. The thermal denaturation is a relatively fast process compared with ionic dissociation. A high molecular weight fraction (3 × M_r = 48,000) was found to melt at 42°C at neutral pH but increased to 54°C at pH 12 where the lysyl side chains are predominantly deprotonated. Furthermore, reconstitution of triple helices appeared to be more readily achieved at high pH. Thus it is concluded that ionic repulsion between side chains causes destabilization of the triple helix and hinders reconstitution.     

Structural changes in 16S RNA from Escherichia coli upon unfolding by urea

The urea-induced unfolding of 16S RNA at low ionic strength has been studied by dynamic light scattering, uv spectroscopy, and some hydrodynamic methods. Three components could be resolved in the photon correlation spectra of scattered light, using the inverse Laplace transform SIPP program [G. R. Danovich and I. N. Serdyuk (1983) in Photon Correlation Techniques in Fluid Mechanics, vol. B38, E. O. Schulz-Dubois, Ed., Springer, Berlin/Heidelberg, New York, p. 315]. One component is assigned to the center-of-mass translation of the RNA, another one to a combination of translational and internal motion, and the last to diffusion of urea clusters. The hydrodynamic dimensions of RNA increase strongly upon transition from 4 to 6M urea. We conclude that up to 2M urea, 16S RNA is highly elongated, and coiled above 4M urea, with a great increase of the hydrodynamic dimensions of RNA being observed upon transition from 4 to 6M urea. A scheme for RNA unfolding is proposed. © 1993 John Wiley & Sons, Inc.     

Affinity labeling of rabbit skeletal muscle phosphorylase kinase by 5′-(p-fluorosulfonylbenzoyl)adenosine

Simple mixing of acid purified histones H₃ and H₄ in equimolar quantities at low ionic strength near pH 7 does not yield the tetramer but rather a high M_r aggregate. Dialysis of acid extracted total or core histones into 2 M NaCl 150 mM phosphate (pH 7.4) followed by fractionation of the histone complexes at lower ionic strength (150 mM NaCl) results in an H₃H₄ tetramer of a structure identical to that derived from salt-extracted histones. Dialysis of acid extracted total or core histones directly into the lower ionic strength buffer with subsequent fractionation, results in H₃H₄ tetramer of closely similar structure.     

pH and salt effects on the slow intermediates of the bacteriorhodopsin photocycle

Purple membrane fragments of Halobacterium halobium were used to investigate pH and salt effects on the kinetics of M ₄₁₂, O ₆₆₀ and BR ₅₆₈. The flash-induced absorbance changes were measured in the 5–9 pH range, at low ionic strength and at 4 M NaCl. The results are consistent with a model which implies a branching in the last part of the bacteriorhodopsin photocycle.     

The Donnan effect in tobacco mosaic virus and its components

Osmotic pressure studies were carried on tobacco mosaic virus (TMV) and its components, protein and RNA, as well as on bis(3,3′-aminopropyl)amine, reported to be present in TMV preparations. Solvents were phosphate and barbital buffers at different values of pH and ionic strength. Measurements were made at room temperature. The Donnan effect was exhibited by TMV protein in phosphate buffer of 0.01 ionic strength at pH values ranging between 5.8 and 7.5. The observed values of the Donnan effect at pH 5.8 and 5.97 were in reasonable agreement with theoretical values calculated from the charge obtained by hydrogen ion titration. TMV-RNA in phosphate buffer at pH 7.5 and ionic strength 0.01 did not exhibit more than 1% of the expected Donnan effect. This is explained tentatively as the result of firm binding of metal ions. Negative values of osmotic pressure were observed with bis(3,3′-aminopropyl)amine. Similar anomalous osmosis was sometimes observed with TMV protein and with TMV. In agreement with earlier observations, TMV did not exhibit the Donnan effect in phosphate buffer of 0.01 ionic strength at pH values ranging from 5.5 to 8.0. However, TMV dialysed extensively in the presence of EDTA at pH 8.5 and TMV produced by reconstitution of purified protein and RNA did exhibit the Donnan effect in both phosphate and barbital buffers. The magnitude was of the same order as that calculated from the net charge determined by hydrogen ion titration. When reconstituted TMV, which did exhibit Donnan effect, was treated with calcium ions, the effect was abolished.     

The structure of a spherical plant virus (bromegrass mosaic virus) established by neutron diffraction.

The localization of the RNA in a spherical plant virus is established by neutron diffraction. For BMV, this localization is different at low pH than it is at high pH (swollen virus).     

A CD study of the role of metal ions in the conformation of poly(L-lysine)

The effect of LiCl, NaCl, and CsCl as univalent salts, and of CaCl₂, ZnCl₂, and MgCl₂ as divalent salts, on the α and antiparallel β-sheet, and random conformations of poly(L-lysine) (PLL), in water at room temperature were examined by means of CD and compared quantitatively on the basis of elliptical strength at the maximal peak. Changes in the α-helical and antiparallel β-sheet helical conformations of PLL were markedly dependent on the salt concentrations of LiCl, NaCl, and CsCl, which induced decreases in negative intensity in that order. The CD spectrum of the random conformation, the most disordered form, displayed positive cotton effect in concentrations of these salts up to 3.0M and a negative peak in concentrations of 6.0M. The effect of these salts on the random conformation of PLL was stronger than that on the α- and β-conformations in higher concentrations. The CD spectrum of the random conformation in the presence of CaCl₂, ZnCl₂, and MgCl₂, on the other hand, showed negative cotton effect in salt concentrations as low as 3.0M. It was impossible, however, to measure the effect on α- and β-conformations of ZnCl₂ and MgCl₂ above concentrations of 10 mM because of a solubility problem with salts in alkaline solution.     

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c

 The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′_IS =(E°′) _{I =0.1 M}–(E°′) _{I =0 M}=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK _a for a residue deprotonation is the key event of this conformational change. Received: 7 April 1999 / Accepted: 19 July 1999     

The control by RNA of pyruvate kinase activity in human skeletal muscle

Summary About 25% of total pyruvate kinase activity in human skeletal muscle is associated with the ribonucleoprotein complexes soluble in salt solutions of high ionic strength. These complexes, called form M_B, crystallize readily from 48% saturated ammonium sulfate at pH 5.6.Crystalline preparations represent a heterogenous population of ribonucleoprotein complexes displaying a graduated activity and a variable RNA content. Free protein was not detected in the preparations.Fractionation of crystalline complexes in salt solutions of varying ionic strength and pH, followed by gel filtration on Sephadex G-200 led to the separation of two nucleoprotein fractions with very high specific activity. Fractions containing 30% RNA and 85% RNA respectively revealed a specific activity of 660–670 U/mg protein at 25°C.Pyruvate kinase form M_A was extracted from muscle homogenate with distilled water, purified to homogeneity and crystallized. It contained less than 0.2% RNA and had a specific activity of 270 U/mg. Active ribonucleoprotein complexes gave in double immunodiffusion test the precipitation bands with the anti-M_A sera at the same protein concentration of both antigens, M_B and M_A.Pyruvate kinase M_B with high activity is sensitive to treatment with RNase. Digestion with RNase for 10 min at 25°C diminished the initial specific activity to about one third. Similar residual activity was found in crystalline ribonucleo protein complexes with low RNA content (3.5–20% RNA) which are resistant to further inactivation by RNase.These results implicate the enhancement and control of pyruvate kinase activity by RNA bound to the enzyme.This work was supported by a grant from the Biochemical and Biophysical Committee of Polish Academy of Sciences.     

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study

The interaction of bovine prothrombin with Ca²⁺ and Mg²⁺ ions was investigated by following H⁺ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca²⁺ and Mg²⁺ binding is characterized by high- and low-affinity sites. M²⁺ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H⁺ release by M²⁺ is bell shaped and consistent with controlling pK_a values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca²⁺ and Mg²⁺ titrations following H⁺ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M²⁺. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca²⁺ and remains dimeric in the presence of Mg²⁺. These results and those for metal ion-dependent H⁺ release indicate that H⁺ release occurs concomitantly with association processes involving prothrombin.Abbreviations GLA -carboxyglutamic acid; fragment 1. amino terminal residues 1–156 of bovine prothrombin - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - PS/PC phosphatidylserine/phosphatidylcholine vesicles - ionic strength     

Ionic strength effects on macroion diffusion and excess light-scattering intensities of short DNA rods

Quasielastic and static light-scattering measurements were made on DNA isolated from chicken erythrocyte mononucleosomes as a function of ionic strength between 6 × 10^?4 and 1.0M. A transition from single-exponential autocorrelation functions to markedly non-single-exponential decays was observed around 10^?2M ionic strength and was accompanied by a large decrease in the excess light-scattering intensity. Autocorrelation functions recorded below 10^?2M salt were well fit by the sum of two exponential relaxation which differed by as much as 100-fold in time constants. Apparent diffusion coefficients for the fast and slow processes plateaued around 10^?3M with numerical values approximately 10-fold and 1/10, respectively, of the translational diffusion coefficient for mononucleosome DNA at high ionic strength. This behavior is similar to that observed with poly(L -lysine), for which the slow decay has been associated with a transition to an extraordinary phase. The strong and complex salt dependence observed here illustrates potential difficulties in deriving structural information from scattering by polyions at low ionic strength.     

Osmotic Pressure of DNA Solutions and Effective Diameter of the Double Helix

Abstract

A simple osmometer with nuclear filters (polymer films with pores of a preset diameter) were used to measure the osmotic pressure of Col El plasmid DNA solutions in the concentration range of 1–4 mg/ml DNA. Linear and open circular DNA forms proved to have the same osmotic pressure within the experimental accuracy. The results of the measurements were used for calculating the second virial coefficient A ₂ of the solution of DNA segments and the effective chain diameter d _eff in the ionic strength range of 10^?2-0.1 M, As the ionic strength is lowered from 0.1 to 10^?2 M the effective diameter of DNA increases from 80 to 220 A. The results are in rather good agreement with theory and with other experimental data.     

Phosphate Activates the Phosphoenolpyruvate Carboxylase from the C4 Plant Amaranthus viridis L.

Phosphoenolpyruvate carboxylase from Amaranthus viridis leaves was activated by inorganic orthophosphate in a concentration- and pH-dependent manner. Maximal activation at pH 7.0 was achieved at phosphate concentrations above 20 mM, and a positive cooperativity was observed for the binding of the anion at this pH. At pH 8.0 the maximum of activity was achieved at 10 mM phosphate; higher concentrations reduced the activation. K_M for phosphoenolpyruvate-Mg at pH 7.0 was lowered by phosphate in all concentrations tested up to 30 mM. While at pH 8.0 the K_M values were lower than that of the control up to 10 mM phosphate; higher anion concentrations raised the minimum value of K_M at this pH. V_MAX increased at pH 7.0, and remained unchanged at pH 8.0. A K_A value of 0.41 mM was calculated for phosphate at the alkaline pH. The phosphate analogue arsenate also behaved as an activating agent, while other anions (e.g. nitrate, nitrite, sulfate, tetraborate) were ineffective. The phosphate-activated enzyme was shown to be insensitive to glucose-6-phosphate, but was inhibited by l -malate to the same extent as the control.     

Studies on the stabilizing forces of simple RNA viruses: II. Stability,dissociation and reassembly of cucumber mosaic virus

The stability properties of cucumber mosaic virus were investigated in relation to those of two other, well-described, icosahedral RNA viruses of similar geometry; the cowpea chlorotic mottle virus and the turnip yellow mosaic virus. High concentrations of neutral salts caused the dissociation of cucumber mosaic virus into its constituent RNA and protein subunits irrespective of the pH of the solution. At low ionic strength the effect of pH on the infectivity and the sedimentation behavior of the virus was tested between pH 4.0 and 8.5. No effect was noticed in this range, but significant change became evident at pH 9.8 and was complete at pH 10.45. The products of this alkaline treatment were a mixture of slower sedimenting nucleoproteins. The RNA inside cucumber mosaic virus was accessible to pancreatic ribonuclease. There was little or no pH-dependence of the ribonuclease susceptibility. Under no circumstances were protein capsids of cucumber mosaic virus ever obtained, neither by degradation of the virion, reassembly of the protein subunits, nor directly from the infected plant. These stability properties of cucumber mosaic virus are strikingly different from those of cowpea chlorotic mottle virus and turnip yellow mosaic virus, as reported in the literature, and indicate the possession of only weak inter-protein subunit linkages, or their total absence.