The observation of the local ordering characteristics of spermidine-condensed DNA: atomic force microscopy and polarizing microscopy studies.

Condensation of DNA by multivalent cations can provide useful insights into the physical factors governing the folding and packaging of DNA in vivo. In this work, local ordered structures of spermidine-DNA complexes prepared from different DNA concentrations have been examined by using atomic force microscopy (AFM) and polarizing microscopy (PM). Two types (I and II) of DNA condensates, significantly different in sizes, were observed. It was found that for extremely dilute solutions (DNA concentrations around 1 ng/microl or below), the DNA molecules would collapse into toroidal structures with a volume equivalent to a single lambda-DNA (type I). In relatively dilute solutions (DNA concentrations between 1 and 10 ng/microll), a significantly larger structure of multimolecular toroids (circular and elliptical, type II) were formed, which were constructed by many fine particles. Measurements show that the average diameter of these fine particles was similar to the outer diameter of the monomolecular toroids observed in extremely dilute solutions, and the thickness of the multimolecular toroids had a distribution of multi-layers with height increments of 11 nm, indicating that the multimolecular toroidal structures have lamellar characteristics. Moreover, by enriching the DNA-spermidine complexes in very diluted solution, branch-like structures constructed by subunits were observed by using AFM. The analysis of the pellets in polarizing microscopy reveals a liquid-crystal-like pattern. These observations suggest that DNA-spermidine condensation could have multiple stages, which are very sensitive to the DNA and spermidine concentrations.     

The mechanism of ATP–G-actin hydrolysis in Mg<Superscript>2+</Superscript>-containing solutions

NMR proton spectra were recorded in the range of proton resonance in the nucleotide aromatic ring of monomeric ATP–G-actin and the Mg²⁺–ATP–G-actin solutions in D2O to study the mechanism of ATP–G-actin hydrolysis and its role in F-actin formation in Mg²⁺-containing solutions. The experimental data show variations in the proton chemical shifts of the H2 and H8 peaks and splitting of the H8 resonance peak of G-actin-bound ATP adenine caused by interaction with magnesium dication. The observed variations in spectra are explained by hydrolysis of monomeric ATP–G-actin to ADP–G-actin, which is regarded as the initial stage of the G-actin to F-actin transformation.     

Membrane potentials in the Donnan equilibrium. II

Measurements were made of electromotive force in the Donnan equilibrium of systems containing dilute solutions of protein and acid. Removal of the membrane produced a decrease of no more than 2 to 4 mv. in electromotive force, while the membrane potentials, as estimated by the usual arbitrary assumption, were of the order of 12 to 34 mv. Ion ratios, as calculated from analyses for total chloride, were definitely greater than those calculated from the electromotive force of cells with salt bridges, as if there had been combination of some of the chloride ion with protein.     

Stable interaction between G-actin and neurofilament light subunit in dopaminergic neurons

Excessive accumulation of neurofilaments in the cell bodies and proximal axons of motor neurons is a major pathological hallmark of motor neuron diseases. In this communication we provide evidence that the neurofilament light subunit (68 kDa) and G-actin are capable of forming a stable interaction. Cytochalasin B, a cytoskeleton disrupting agent that interrupts actin-based microfilaments, caused aggregation of neurofilaments in cultured mesencephalic dopaminergic neurons, suggesting a possible interaction between neurofilaments and actin; which was tested further by using crosslinking reaction and affinity chromatography techniques. In the cross-linking experiment, G-actin interacted with individual neurofilament subunits and covalently cross-linked disuccinimidyl suberate, a homobifunctional cross-linking reagent. Furthermore, G-actin was extensively cross-linked to the light neurofilament subunit with this reagent. The other two neurofilament subunits showed no cross-linking to G-actin. Moreover, neurofilament subunits were retained on a G-actin coupled affinity column and were eluted from this column by increasing salt concentration. All three neurofilament subunits became bound to the G-actin affinity column. However, a portion of the 160 and 200 kDa neurofilament subunits did not bind to the column, and the remainder of these two subunits eluted prior to the 68 kDa subunit, suggesting that the light subunit exhibited the highest affinity for G-actin. Moreover, neurofilaments demonstrated little or no binding to F-actin coupled affinity columns. The phosphorylation of neurofilament proteins with protein kinase C reduced its cross-linking to G-actin. The results of these studies are interpreted to suggest that the interaction between neurofilaments and actin, regulated by neurofilament phosphorylation, may play a role in maintaining the structure and hence the function of dopaminergic neurons in culture.     

Interactions of myosin subfragment 1 isozymes with G-actin

The polymerization of G-actin by myosin subfragment 1 (S-1) isozymes, S-1(A1) and S-1(A2), and their proteolytically cleaved forms was studied by light-scattering, fluorescence, and analytical ultracentrifugation techniques. As reported previously, S-1(A1) polymerized G-actin rapidly while S-1(A2) could hardly promote the assembly reaction (Chaussepied & Kasprzak, 1989a; Chen and Reisler, 1990). This difference between the isozymes of S-1 was traced to the very poor, if any, ability of G-actin-S-1(A2) complexes to nucleate the assembly of actin filaments. The formation of G-actin-S-1(A2) complexes was verified in sedimentation velocity experiments and by fluorescence measurements using pyrene-labeled actin. The G-actin-S-1(A2) complexes supported the growth of actin filaments and accelerated the polymerization of actin in solutions seeded with MgCl2-, KCl-, and S-1(A1)-generated nuclei. The growth rates of actin-S-1(A2) filaments were markedly slower than those for actin-S-1(A1) filaments. Proteolytic cleavage of S-1 isozymes at the 50/20-kDa junction of the heavy chain greatly decreased their binding to G-actin and thus inhibited the polymerization of actin by S-1(A1). These results are discussed in the context of G-actin-S-1 interactions.     

The State of Water in Muscle Tissue as Determined by Proton Nuclear Magnetic Resonance

The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T₁ and T₂, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T₁ and T₂ in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized.     

Comparison of ultrafiltration systems for concentration of biologicals

Ultrafiltration has been used with increasing frequency in recent years in biological laboratories for concentration, separation or purification of biological material. No data have been available on the comparison of the characteristics of Ultrafiltration systems currently in use. This study compares the filtration characteristics of four systems using commercially available membranes on suspensions and solutions: suspension of protein micelles (casein), cell debris (E. coli) and catalase solution. None of the four systems considered is found to be generally superior for all the suspensions and solutions. Vibration systems were most effective when relatively large particles were involved, while laminar flow recycling systems with high wall shear rates were best for dilute suspensions and proteins in solution. It was found that shearing inactivates enzymes in both recycle and vibration systems. It was also observed that vibration actually reduces flux in dilute solutions.     

Quantitative analysis of G-actin transport in motile cells

Cell migration is based on an actin treadmill, which in turn depends on recycling of G-actin across the cell, from the rear where F-actin disassembles, to the front, where F-actin polymerizes. To analyze the rates of the actin transport, we used the Virtual Cell software to solve the diffusion-drift-reaction equations for the G-actin concentration in a realistic three-dimensional geometry of the motile cell. Numerical solutions demonstrate that F-actin disassembly at the cell rear and assembly at the front, along with diffusion, establish a G-actin gradient that transports G-actin forward “globally” across the lamellipod. Alternatively, if the F-actin assembly and disassembly are distributed throughout the lamellipod, F-/G-actin turnover is local, and diffusion plays little role. Chemical reactions and/or convective flow of cytoplasm of plausible magnitude affect the transport very little. Spatial distribution of G-actin is smooth and not sensitive to F-actin density fluctuations. Finally, we conclude that the cell body volume slows characteristic diffusion-related relaxation time in motile cell from ∼10 to ∼100 s. We discuss biological implications of the local and global regimes of the G-actin transport.     

Differences in internal dynamics of actin under different structural states detected by neutron scattering

F-actin, a helical polymer formed by polymerization of the monomers (G-actin), plays crucial roles in various aspects of cell motility. Flexibility of F-actin has been suggested to be important for such a variety of functions. Understanding the flexibility of F-actin requires characterization of a hierarchy of dynamical properties, from internal dynamics of the actin monomers through domain motions within the monomers and relative motions between the monomers within F-actin to large-scale motions of F-actin as a whole. As a first step toward this ultimate purpose, we carried out elastic incoherent neutron scattering experiments on powders of F-actin and G-actin hydrated with D₂O and characterized the internal dynamics of F-actin and G-actin. Well established techniques and analysis enabled the extraction of mean-square displacements and their temperature dependence in F-actin and in G-actin. An effective force constant analysis with a model consisting of three energy states showed that two dynamical transitions occur at ∼150 K and ∼245 K, the former of which corresponds to the onset of anharmonic motions and the latter of which couples with the transition of hydration water. It is shown that behavior of the mean-square displacements is different between G-actin and F-actin, such that G-actin is “softer” than F-actin. The differences in the internal dynamics are detected for the first time between the different structural states (the monomeric state and the polymerized state). The different behavior observed is ascribed to the differences in dynamical heterogeneity between F-actin and G-actin. Based on structural data, the assignment of the differences observed in the two samples to dynamics of specific loop regions involved in the polymerization of G-actin into F-actin is proposed.     

The toxofilin-actin-PP2C complex of Toxoplasma: identification of interacting domains

Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure-function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein-protein interactions that are important to parasite motility.     

Structural aspects of skeletal muscle G-actin molecule as studied by proteolytic digestion: effect of nucleotide

Trypsin and chymotrypsin were used as probes of conformation of G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. G-actin is known to be nonrefractory to proteolysis [Jacobson, G.R., and Rosenbusch, J.P. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2742-2746]. It is really true that G-actin is cut easily into a 33-kDa fragment by trypsin or chymotrypsin, but only when free ATP is present in the medium. After the removal of free ATP from the medium, G-actin became more refractory to proteolysis. The amounts of degradation of G-actin depended on the ATP concentration in the medium with saturating at about 0.5 mM. epsilon-ADP also had the effect and its fluorescence spectrum was changed on the addition of G-actin. After the removal of free ATP, G-actin still bound 1 mol/mol of ATP. So, the present results suggest the presence of a second ATP interaction site on G-actin and that ATP interaction at this site induces conformational changes in G-actin molecule.     

Interpretation of fluorescence correlation spectra of biopolymer solutions

Fluorescence correlation spectroscopy (FCS) is regularly used to study diffusion in non‐dilute “crowded” biopolymer solutions, including the interior of living cells. For fluorophores in dilute solution, the relationship between the FCS spectrum G(t) and the diffusion coefficient D is well‐established. However, the dilute‐solution relationship between G(t) and D has sometimes been used to interpret FCS spectra of fluorophores in non‐dilute solutions. Unfortunately, the relationship used to interpret FCS spectra in dilute solutions relies on an assumption that is not always correct in non‐dilute solutions. This paper obtains the correct form for interpreting FCS spectra of non‐dilute solutions, writing G(t) in terms of the statistical properties of the fluorophore motions. Approaches for applying this form are discussed. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 260–266, 2016.     

The effect of simple shear flow on the helix–coil transition of poly‐L‐lysine

Studies of the helix‐to‐coil transition in dilute solutions of poly‐L ‐lysine, dissolved in mixtures of water and methanol (MeOH), have been carried under shear flow using flow birefringence and modulated polarimetry. The fraction of helical conformations in a given solution remains independent of shear rate for MeOH concentrations above and below the critical value for the helix‐coil transition (i.e., 87.5% MeOH). For the 87.5% MeOH solutions, a shear‐induced helix‐to‐“stretched” coil transition occurs above a critical shear rate. Induction times for the transition show a temperature and shear rate dependence that can be described in terms of an activated jump process. Measurements of circular birefringence on cessation of flow also show that the transition is reversible, with the stretched coil reverting to the helical state on a time scale of several seconds. The activation energy for the jump process is found to be 16.2 kJ/mole. © 1999 John Wiley & Sons, Inc. Biopoly 50: 589–594, 1999     

Viscosity study of interactions between sodium alginate and CTAB in dilute solutions at different pH values

Interactions between anionic polyelectrolyte sodium alginate and the cationic surfactant cetytrimethylammonium bromide (CTAB) have been investigated by viscosity measurement techniques. The polymer–surfactant interactions are observed between alginate and CTAB at different pH in dilute solution. The results show that the rheological response of alginate dilute solutions is sensitive to a change of pH in the low pH range. The steady shear and intrinsic viscosity measurements reveal that the strong association between alginate and CTAB by electrostatic attraction above pH 5.0. However, as the pH value of solution decrease from 5.0 to 3.0, the strong association between alginate and CTAB is affected by not only electrostatic attraction but also hydrophobic interaction.     

Maleimidobenzoyl-G-actin: structural properties and interaction with skeletal myosin subfragment-1

We have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt--and myosin subfragment 1 (S-1)-induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain (Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032). The far-ultraviolet CD spectrum and alpha-helix content of the MBS-actin were identical with those displayed by native G-actin. 45Ca2+ measurements showed the same content of tightly bound Ca2+ in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl + 5 mM MgCl2 led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6 mg/mL, at 25 degrees C, pH 8.0. The MBS-F-actin formed activated the Mg2(+)-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was not to be at all affected by its specific covalent conjugation to S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochemical dopamine visualization as a method for estimation of globular actin content in cytosol of living cells

The possibility to reveal globular (G-) actin in cell cytosole by means of microscopy has been studied. The applicability of this method was in particular evaluated for diagnostics of malignant cells, whose main pathocytological feature is an anomalously high content of G-actin in cytosol. The cells of a common origin but with different states of cytosolic actin were analyzed by means of cytochemical reaction for biogenic amines using Falck-Hillarp method after 40-h incubation of the cells in dopamine-containing cultivation medium. Mouse embryo cell line BALB/3T3, clone A31 with differentiated actin cytoskeleton were used as a control cell line. The same cells infected with pathogenic virus SV-40 (cell line 3T3B-SV40) exhibited a malignant phenotype; their cytosol mainly consisted of G-actin. Manifold increase in fluorescence intensity of cytosol and karyoplasms, the loci with the highest G-actin concentration, was revealed in malignant cells in comparison with their healthy prototype. Thus, it was shown that G-actin of malignant cells is a diagnostic target for dopamine, which, as it was earlier shown, penetrates into the cytosol, polymerizes G-actin, incorporating into the filaments as integral component in the 100:1 ratio, and thus fluorescently labels G-actin due to conversion into isoquinoline by reaction with formaldehyde. Besides, dopamine exhibited a strong cytotoxicity that considerably reduced the viability of malignant cells. The data suggest that the content of Gactin in cytosol of living cells can be quantitatively estimated by fluorescence intensity of cytosol following incubation of the cells in dopamine-containing medium.     

Stability of helix-rich proteins at high concentrations

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions.     

Measurement of free space and sorption of large molecules by cereal roots

Abstract. Large molecular weight solutes that do not penetrate the root have been used to correct for the surface film in measurements with mannitol of the volume of the Apparent Free Space (FS) in bailey roots. The results are compared with those obtained using other correction techniques for elimination of the surface film. Large molecules seem to be adsorbed on the root surface and the kinetics of adsorption differ between the polyhydric alcohol mannitol or the polysaccharide dextran on the one hand, and the polyether polyethylene glycol (PEG-4000) on the other. The significance of this difference in kinetics is discussed in relation to the use of PEG as an osmoticum in studies on root water relations and its effect on ion uptake. Although smaller molecular weight PEG's penetrate the FS and diminish sodium uptake from 10 mol m⁻³ NaCl, more dilute solutions of mannitol and larger PEG polymers are unlikely to affect ion uptake from dilute nutrient solutions. Use of these substances along with labelled nutrients in kinetic studies of the compartmentation of ions in roots can help to distinguish between ions associated with the surface film, those in the FS and those that have crossed the cell membranes into the protoplast.     

Detection of conformational changes in actin by proteolytic digestion: Evidence for a new monomeric species

When KCl is added to a solution of G-actin to induce full polymerization, a decrease in the rate at which actin undergoes enzymatic proteolysis occurs. This decrease cannot be accounted for by factors affecting the enzymes employed, but rather appears to be due to a change in the conformation of G-actin. Partially polymerized actin solutions also show a reduction in digestibility which is dependent on the F-actin content, suggesting that F-actin is essentially indigestible. Moreover, low rates of digestion were also observed at sub-critical actin concentrations, where actin in the presence of 0.1 m-KCl does not polymerize. This indicates that a confomational change occurs in G-actin before the polymerization step.At sub-critical concentrations in 0.1 m-KCl, actin is in a truly monomeric state as judged by its viscosity characteristics, its inability to enhance the rate of polymerization of G-actin and its possession of ATP as the actin-bound nucleotide. These data support the existence of a new species of actin, called F-ATP-actin monomer, which has the same physical properties and the same bound nucleotide as G-actin, but digestion characteristics like F-actin. Since F-ATP-actin monomers have the same low susceptibility to proteolysis as F-ADP-actin polymers, and because both G-ATP-actin and G-ADP-actin have similar high rates of digestion, the observed change in the conformation of actin cannot be due to the phosphorylated state of the actin-bound nucleotide. Instead, the conformational change appears to be caused by the addition of KCl to G-actin.The newly-detected monomeric species is considered to be an intermediate in the polymerization process where F-ATP-actin monomers form a population of polymerizable molecules which must reach a critical concentration before nucleation and F-actin polymer formation begin.     

Mapping the cofilin binding site on yeast G-actin by chemical cross-linking

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the α3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 Å) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.