Vitellogenic ovarian follicles of Drosophila exhibit a charge-dependent distribution of endogenous soluble proteins

In ovarian follicles of Drosophila, soluble endogenous charged proteins are asymmetrically distributed dependent upon their ionic charge. Reversal of the normal ionic difference across the intercellular bridges which connect nurse cells to their oocyte results in a redistribution of these proteins. Twelve soluble endogenous acidic proteins were identified by 2-D gel electrophoresis as being present in both oocytes and nurse cells in samples run on four or more gels. Of these, following osmotically induced reversal of the electrical transbridge gradient the concentration of seven proteins decreased in the oocyte while nurse cell concentrations of all twelve proteins increased. Of seven basic proteins analyzed, following reversal of the electrical gradient the concentration of all seven increased in oocytes. Four of these decreased in nurse cells, while nurse cell concentrations of the remaining three basic proteins also appeared to decrease, but yielded spots too faint for measurement. Data presented here demonstrate that, as in the Saturniidae, the ionic gradient across the nurse cell-oocyte intercellular bridges of the dipteran, Drosophila, can influence the distribution of soluble endogenous charged molecules.     

On the Stability of the Soluble Amyloid Aggregates

Many amyloid proteins form metastable soluble aggregates (or protofibrils, or protein nanoparticles, with characteristic sizes from ∼10 to a few hundred nm). These can coexist with protein monomers and amyloid precipitates. These soluble aggregates are key determinants of the toxicity of these proteins. It is therefore imperative to understand the physical basis underlying their stability. Simple nucleation theory, typically applied to explain the kinetics of amyloid precipitation, fails to predict such intermediate stable states. We examine stable nanoparticles formed by the Alzheimer's amyloid-β peptide (40 and 42 residues), and by the protein barstar. These molecules have different hydrophobicities, and therefore have different short-range attractive interactions between the molecules. We also vary the pH and the ionic strength of the solution to tune the long-range electrostatic repulsion between them. In all the cases, we find that increased long-range repulsion results in smaller stable nanoparticles, whereas increased hydrophobicity produces the opposite result. Our results agree with a charged-colloid type of model for these particles, which asserts that growth-arrested colloid particles can result from a competition between short-range attraction and long-range repulsion. The nanoparticle size varies superlinearly with the ionic strength, possibly indicating a transition from an isotropic to a linear mode of growth. Our results provide a framework for understanding the stability and growth of toxic amyloid nanoparticles, and provide cues for designing effective destabilizing agents.     

Ionic regulation of proteins

Ion binding modulates the structural and the functional properties of several proteins. The molecular bases of such interactions depend on the charge density of ions, in turn influencing their ability to bind water molecules. The broad range of proteins submitted to ionic control, makes it interesting a general evaluation of the role played by ions in the homeostasis of intra and extracellular compartments and in a number of physio-pathological conditions, with special attention to the coagulation cascade and cryoglobulin aggregation.     

Effects of allapinine on sodium currents in neurons isolated from the rat trigeminal ganglion and cardiomyocytes

The effects were investigated of allapinine, diterpene alkaloid on ionic currents in voltage-clamped trigeminal neurons and cardiomyocytes isolated from rats. Allapinine application was found to exert an inhibitory effect on inward tetrodotoxin-sensitive sodium currents without changing their voltage dependence. Potential differences between the mechanisms of antiarrhythmic action of diterpene alkaloids and standard antiarrhythmic substances are examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Plant Chemistry, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 201–206, March–April, 1990.     

A cure for traffic jams: small molecule chaperones in the endoplasmic reticulum

Folding in the endoplasmic reticulum is the limiting step for the biogenesis of most secretory pathway cargo proteins; proteins which fail to fold are initially retained in the endoplasmic reticulum and subsequently often degraded. Mutations that affect secretory protein folding have profound phenotypes irrespective of their direct impact on protein function, because they prevent secretory proteins from reaching their final destination. When unicellular organisms are stressed by fluctuation of temperature or ionic strength, they synthesize high concentrations of small molecules such as trehalose or glycerol to prevent protein denaturation. These osmolytes can also stabilize mutant secretory proteins and allow them to pass secretory protein quality control in the endoplasmic reticulum. Specific ligands and cofactors such as ions, sugars, or peptides have similar effects on specific defective proteins and are beginning to be used as therapeutic agents for protein trafficking diseases.     

Effect of nonachlazine on transmembrane ionic currents in the frog atrial trabeculae

The effect of the antianginal drug nonachlazine displaying antiarrhythmic properties on transmembrane ionic currents in the frog atrial fibers was studied in experiments on isolated trabeculae of the frog atria. The transmembrane ionic currents were measured by a voltage clamp technique based on a double sucrose gap arrangement. Nonachlazine (1.03 X 10(-5) mol/l) decreased the amplitude of the fast inward current whatever the magnitude of membrane potential. The drug inhibited the slow inward current and prevented the adrenaline-increased permeability of the slow sodium-calcium channel if external sodium ions were replaced by choline chloride. Nonachlazine (1.03 X 10(-5) mol/l) diminished the amplitude of the inward ionic current in a calcium-free medium as well. The stimulatory effect of prostacycline (2 X 10(-7) mol/l) on the fast inward ionic current was inhibited by nonachlazine. The data obtained suggest that the antiarrhythmic effect of nonachlazine might be linked with the inhibition of the fast sodium inward current and the slow calcium inward current.     

Interaction between non-histone chromatin protein HMGB1 and linker histone H1

The fundamental possibility of interaction between non-histone chromatin protein HMGB1 and linker histone H1 was studied in the solutions with different ionic strength by intrinsic UV-fluorescence, far and near-UV CD and spectrophotometry. The obtained data allow us to assume that the increase of histone H1 content in the HMGB1 solutions in a low ionic strength is accompanied by the destruction of HMGB1 associates. The interaction between proteins of HMGB1 and H1 causes the increase in the number of ordered regions in the protein molecules and the minor changes in their tertiary structure.     

Detergents as tools in membrane biochemistry.

Detergents are invaluable tools for studying membrane proteins. However, these deceptively simple, amphipathic molecules exhibit complex behavior when they self-associate and interact with other molecules. The phase behavior and assembled structures of detergents are markedly influenced not only by their unique chemical and physical properties but also by concentration, ionic conditions, and the presence of other lipids and proteins. In this minireview, we discuss the various aggregate forms detergents assume and some misconceptions about their structure. The distinction between detergents and the membrane lipids that they may (or may not) replace is emphasized in the most recent high resolution structures of membrane proteins. Detergents are clearly friends and foes, but with the knowledge of how they work, we can use the increasing variety of detergents to our advantage.     

The effects of ionic strength on the protein conformation and the fluidity of porcine intestinal brush border membranes. Fluorometric studies using N-[7-dimethylamino-4-methylcoumarinyl]maleimide and pyrene

The effects of ionic strength on the conformation around the SH groups of the proteins and the lipid fluidity of porcine intestinal brush border membranes were studied using two fluorescent dyes, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM) and pyrene. The extent of DACM labeling to the SH groups of the membrane proteins was accelerated depending on the KCl concentrations in medium. A quenching study of DACM-labeled membranes with acrylamide showed that the proximity of the quencher to the fluorescence-labeled SH groups in the membrane proteins is increased with increasing ionic strength of medium. An implication of the conformational changes around SH groups in the membrane proteins with increase of ionic strength was also obtained from the stimulation of guanidine effect on the fluorescence parameters of DACM-labeled membranes by addition of KCl. On the other hand, the results of the quenching study with KI, excimer fluorescence, and polarization measurements of pyrene-labeled membranes suggested an increase of membrane fluidity on addition of KCl to medium. The temperature dependence of polarization of the complex strongly suggested that the rotational freedom of pyrene molecules embedded into the lipid layers of the membranes is increased by addition of KCl. In fact, the harmonic means of the rotational relaxation times of pyrene molecules in the membranes with and without 100 mM KCl were estimated to be about 2900 and 9000 ns at 25 degrees C, respectively. Based on these results, the salt-induced alterations of the conformation in the vicinity of the bound dyes of the membrane proteins and of the membrane fluidity are discussed.     

Interaction of histone H1 molecules in a solution

Molecules of histones H1 isolated from the calf thymus, carp testicles and spermatozoa as well as trypsin-stable fragments of these proteins have been studied from the standpoint of their structure and interaction using methods of differential spectrophotometry, gel filtration and turbidimetry. The globular structure of histone H1 of the calf thymus is formed with an increase in the ionic strength of the medium and it is eluted as dimer with gel chromatography. With a considerable local increase of ionic strength (by addition of NaCl crystals) molecules of histones H1 form high-molecular aggregates from all the studied tissues. This aggregation is a result of interaction of globular trypsin-stable sites. Molecules of histone H1 from carp testicles and spermatozoa as well as their trypsin-stable fragments revealed no differences in the ability to form dimers and aggregates.     

Protein adsorption on histidyl-aminohexyl-Sepharose 4B: II. Application to the negative one-step affinity purification of human β2-microglobulin and Immunoglobulin G

The adsorption of two human proteins, β2-microglobulin and Immunoglobulin G, from uremic patient’s blood ultrafiltrate and plasma, respectively, was investigated on the histidyl-aminohexyl-Sepharose 4B adsorbent. Both target proteins could be adsorbed on the gel through a low affinity for immobilized histidine ligand. However, a fine adjustment of the operating conditions (ionic strength, buffer, pH) prevented their adsorption and thus allowed their “negative affinity” purification (purity estimated by silver nitrate SDS–PAGE) by the removal of the contaminating proteins. This simple and efficient method provides purification under gentle chromatographic conditions and a further characterization of both molecules.     

Sedimentation equilibrium studies on the interaction between cytochrome c and cytochrome c peroxidase

The interaction between cytochrome c and cytochrome c peroxidase was investigated using sedimentation equilibrium at pH 6,20 degrees C, in a number of buffer systems varying in ionic strength between 1 and 100 mM. Between 10 and 100 mM ionic strengths, the sedimentation of the individual proteins was essentially ideal, and sedimentation equilibrium experiments on mixtures of the two proteins were analyzed assuming ideal solution behavior. Analysis of the distribution of mixtures of cytochrome c and cytochrome c peroxidase in the ultracentrifuge cell based on a model involving the formation of a 1:1 cytochrome c-cytochrome c peroxidase complex gave values of the equilibrium dissociation constant ranging from 2.3 +/- 2.7 microM at 10 mM ionic strength to infinity (no detectable interaction) at 100 mM ionic strength. Attempts to determine the presence of complexes involving two cytochrome c molecules bound to cytochrome c peroxidase were inconclusive.     

Discrimination among individual Watson-Crick base pairs at the termini of single DNA hairpin molecules

Nanoscale α-hemolysin pores can be used to analyze individual DNA or RNA molecules. Serial examination of hundreds to thousands of molecules per minute is possible using ionic current impedance as the measured property. In a recent report, we showed that a nanopore device coupled with machine learning algorithms could automatically discriminate among the four combinations of Watson–Crick base pairs and their orientations at the ends of individual DNA hairpin molecules. Here we use kinetic analysis to demonstrate that ionic current signatures caused by these hairpin molecules depend on the number of hydrogen bonds within the terminal base pair, stacking between the terminal base pair and its nearest neighbor, and 5′ versus 3′ orientation of the terminal bases independent of their nearest neighbors. This report constitutes evidence that single Watson–Crick base pairs can be identified within individual unmodified DNA hairpin molecules based on their dynamic behavior in a nanoscale pore.     

Cardiac Myosin Filaments are Maintained by Stochastic Protein Replacement

Myosin and myosin-binding protein C are exquisitely organized into giant filamentous macromolecular complexes within cardiac muscle sarcomeres, yet these proteins must be continually replaced to maintain contractile fidelity. The overall hypothesis that myosin filament structure is dynamic and allows for the stochastic replacement of individual components was tested in vivo, using a combination of mass spectrometry– and fluorescence-based proteomic techniques. Adult mice were fed a diet that marked all newly synthesized proteins with a stable isotope-labeled amino acid. The abundance of unlabeled and labeled proteins was quantified by high-resolution mass spectrometry over an 8-week period. The rates of change in the abundance of these proteins were well described by analytical models in which protein synthesis defined stoichiometry and protein degradation was governed by the stochastic selection of individual molecules. To test whether the whole myosin filaments or the individual components were selected for replacement, cardiac muscle was chemically skinned to remove the cellular membrane and myosin filaments were solubilized with ionic solutions. The composition of the filamentous and soluble fractions was quantified by mass spectrometry, and filament depolymerization was visualized by real-time fluorescence microscopy. Myosin molecules were preferentially extracted from ends of the filaments in the presence of the ionic solutions, and there was only a slight bias in the abundance of unlabeled molecules toward the innermost region on the myosin filaments. These data demonstrate for the first time that the newly synthesized myosin and myosin-binding protein C molecules are randomly mixed into preexisting thick filaments in vivo and the rate of mixing may not be equivalent along the length of the thick filament. These data collectively support a new model of cardiac myosin filament structure, with the filaments being dynamic macromolecular assemblies that allow for replacement of their components, rather than rigid bodies.     

Invisibility of tissues and the mother/fetus paradox: an hypothesis

How do tissues hide themselves from the body's natural defense system? Anonymity is difficult to sustain in the blood stream. Almost all surfaces become tagged by polyionic molecules, such as proteins, within milliseconds of being exposed to plasma (which, after all, is an abundant source of animal glue). Both the tagging and the gluing are spontaneous events and result from the release of electrostatically bound water when ionic groups of the surface and the protein mutually satisfy their respective charges. The driving forces responsible for the spontaneity of the adsorption/gluing process are various attractive forces and the disordering of bound water leading to a gain in entropy. But suppose the surface of an intruder had no surface charges, causing it to resemble the surface of a slice of (bulk) water. The polyionic proteins would not find anything to tag, there is no entropy gain, there are no attractive interactions, and the interloper would be invisible.     

Quantitative determination of zwitterionic detergents using salt-induced phase separation of Triton X-100

Zwitterionic detergents interfere with the salt-induced phase separation for nonionic detergents in a concentration-dependent manner by shifting the normal cloud point of nonionic detergents to a higher ionic strength at room temperature. This phenomenon was used to determine the concentration of the zwitterionic detergents CHAPS, CHAPSO, and sulfobetaine SB-12 in solution by titration with ammonium sulfate in the presence of Triton X-100. Among the ionic detergents tested, the method was only applicable to sodium cholate. The assay can be used to control the removal of zwitterionic detergents during the reconstitution of membrane proteins in liposomes. However, it cannot be used to determine the specific binding of zwitterionic detergents to highly diluted, pure membrane proteins because of the limited sensitivity. Neither proteins nor phospholipids interfered with this method at concentrations up to 20 mg/ml of test solution (human serum albumin) or 10 mg/ml (phospholipids), respectively. Since the assay is based on the competition between salts and nonionic detergents for water molecules, it is important to equalize the ionic strength of samples and calibration standards.     

Conformation of the ribosomal protein S1 of Thermus thermophilus in solution under different ionic conditions

The structure of protein SI of Thermus thermophilus (M = 61 kDa) in solution at low and moderate ionic strengths (0 M and 100 mM NaCl, respectively) has been studied by small-angle X-ray and neutron scattering. It was found that protein S1 has a globular conformation under both ionic conditions. The modelling of different packing of six homologous domains of S1 on the basis of the NMR-resolved structure of one domain showed that the best fit of calculated scattering patterns from such complexes to experimental ones is observed at a compact package of the domains. The calculated value of the radius of gyration of the models is 28-29 angtroms, which is characteristic for globular proteins with a molecular mass of about 60 kDa. It was found that protein S1 has a tendency to form associates, and the type of the associate depends on ionic strength. These associates have, in general, two or three monomers at a moderate ionic strength, while at a low ionic strength the number of monomers exceeds three and they are packed in a compact manner. Strongly elongated associates were observed in neutron experiments at a moderate ionic strength in heavy water. The association of protein molecules was also confirmed by the data of dynamic light scattering. From these data, the translational diffusion coefficient of protein S1 at a moderate ionic strength was calculated to be (D20,w = (2.7 +/- 0.1) x 10(-7)cm2/s). This value is essentially smaller than the expected value (D20,w = (5.8 - 6.0) x 10(-7)cm2/s) for the S1 monomer in the globular conformation, indicating the association of protein molecules under equilibrium conditions.     

Smart hydrogels for bioseparation

Smart hydrogels are hydrogels which alter their dimension (i.e., either swell or shrink) dramatically upon a small change in an environmental condition, such as temperature, pH, ionic strength, salt type, solvent, etc. Due to large changes in the swelling ratio, the smart hydrogels have been used widely in the separation of various molecules including proteins. Bioseparation using smart hydrogels is convenient, cost effective, and operable in mild conditions. The use of mild conditions during separation is critical for proteins which can be easily denatured or degraded. Smart hydrogels currently used in bioseparation and their limitations as well as improvements to be made are described here. This revised version was published online in July 2006 with corrections to the Cover Date.     

Studies on the stability of the higher-order structure of rat liver chromatin containing high-mobility-group proteins

The stability of the higher-order structure of chromatin containing high-mobility-group (HMG) proteins has been studied in rat liver nuclei by mild micrococcal nuclease digestion at low temperature and fractionation by sucrose gradient centrifugation. Nuclei preparation and digestion, chromatin solubilization and analysis have been carried out in two ionic conditions, 140 mM and 40 mM monovalent cation concentration, avoiding drastic changes in ionic conditions and temperature during preparation and analysis. During the time course of digestion at 140 mM ionic strength a material stable at 80 S appears, whose DNA is cleaved at values around 12 nucleosomes. The distribution of HMG proteins in different chromatin fractions was analyzed by immunodot using antibodies elicited against proteins HMG-1, HMG-2, and HMG-14 and 17. It appears that these proteins have a distribution distinctly different from the bulk of chromatin. They are never found in the chromatin fragments that keep their internucleosomal interactions, indicating that these proteins tend to accumulate in points where the chromatin has a less stable structure.     

Mobility of adsorbed proteins: a Brownian dynamics study

We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies.