EMSA and Single-Molecule Force Spectroscopy Study of Interactions between Bacillus subtilis Single-Stranded DNA-Binding Protein and Single-Stranded DNA

We determined the pKa values of acidic residues in hen lysozyme by comparing the pH dependency of stability between wild type and mutant lysozymes in which a negative charge is eliminated. In the comparison of the stability between wild type and a mutant lysozyme, the difference in pH titration curve between them could be expressed as a two-state process involving protonation of a single acidic residue. The results strongly indicated that the Aune and Tanford theory of protein denaturation [Aune, K.C. and Tanford, C. (1969) Biochemistry 8, 4579-4585] is applicable to protein stability in solution. On the other hand, the pKa values of acidic residues in the presence of low (5 mM) or high (400 mM) salt concentration were determined by means of two-dimensional NMR. We found that the pKa values obtained from the pH dependency of stability were close to those from the NMR experiment under the high salt condition. Moreover, by comparing pKa values at high salt and low salt concentrations, we could evaluate the dependency of two electrostatic interactions (salt bridge and charge-helix dipole interaction) on salt concentration.

The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome. In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions. Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species. Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions. The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices. These results support a model in which bacterioopsin is produced in H. volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally. According to this model, membrane insertion is directed by the seventh helix.

The effect of salt type and concentration on the transformation of an oriented crystalline collagen tendon into a crosslinked network under conditions of equilibrium swelling was investigated. Our main observations are the following. The degree of swelling of crystalline tendons increases at low salt concentration Cs, and decreases at higher Cs for a wide variety of salts. The observation is not reconcilable with swelling taking place in interfibrillar spaces or structural voids. Within the tropocollagen units and at their ends, regions of reduced organization are postulated (as suggested by Bear and by Schmitt) which are able to interact with the diluent in the amorphous‐like manner. At least four different factors should be considered in assessing the role of salt and salt concentration on the shrinkage temperature Ts under isoelectric conditions. They are: (1) specific effects, (2) diluent effects, (3) crosslinking effects, and (4) nonequilibrium effects. The diluent effects are correlated with the salting‐in–salting‐out power of the ions which was characterized in Part I of this series. Smaller amounts of diluents are generally available to the tendon when the salt has a higher salting‐out power, and this corresponds to higher shrinkage temperatures, other conditions being the same. The crosslinking effect raises Ts due to a reduction of the diluent content and, probably for p‐benzoquinone and formaldehyde, also to a reduction of the conformational entropy in the molten state. Nonequilibrium effects arise from the fact that shrinkage and recrystallization are kinetically hindered when the tendon is highly deswollen in strong salting‐out solutions, or when the salt has a crosslinking power. The specific effect is the only effect which is not related to the amount of diluent present in the tendon. Its origin is less clear. For anions such as Cl− and SCN−, it is possibly related to an ability of the ion to prevent intersegmental hydrogen bonding and water carbonyl bridges. The competition of several of the above effects for a given salt solution makes possible various types of dependence of Ts upon Cs: Ts may either continuously decrease or continuously increase with increasing Cs, or it may go through a minimum. In absence of salt, the cooperative character of the transition at the pH at which maximum swelling occurs appears extremely reduced. The large swelling maintains the tendon in the elongated state and this simulates a continuous decrease of Ts on lowering pH. In presence of small quantities of salt, which reduce swelling, the transition is sharp and Ts is decreased with pH up to pH 2, when maximum swelling occurs, and then reincreases on further lowering of the pH. The dependence of Ts upon Cs is more complex than under isoelectric conditions. There is generally an increase of Ts with Cs which is equivalent to an increase of the denaturation temperature with Cs for helical polyelectrolytes in solution. At higher salt concentrations, however, Ts may decrease again, and possibly increase again at still higher salt concentrations, depending upon the effect of the salt solution in the isoelectric zone.

Double‐stranded RNAs (dsRNA) such as poly(I) · poly(C) inhibit protein synthesis in reticulocyte lysate incubated with low concentrations of potassium salts more than in lysate incubated with higher concentration of salt. The inhibition of protein synthesis by dsRNA takes place after an initial lag. In the presence of high salt concentrations the lag period before the inhibition occurs is considerably increased. The maximum inhibitory concentration of poly(I) · poly(C) remains the same at all salt concentrations tested; high concentrations of dsRNA are not inhibitory under different ionic conditions.The inhibition of protein synthesis by dsRNA is due to a loss of the Met‐tRNAf binding activity of native 40 S ribosomal subunits. However, lysates incubated with dsRNA at high salt concentrations retain Met‐tRNAf binding activity. Initiation factors(elF) isolated from ribosomes of lysates incubated under different ionic conditions have been used to determine the activity of the initiation factor, elF‐2, which catalyzes the binding of Met‐tRNAf to 40‐S subunits. The initiation factors from lysates incubated with poly(I) · poly(C) at low salt concentrations are less active in binding Met‐tRNAf and GTP than those from lysates incubated at high salt concentrations. Ribosomes washed with 0.5 M KCl obtained from these lysates are equally active in binding Met‐tRNAf to 40‐S subunits when supplemented with fresh initiation factors, indicating that the loss in Met‐tRNAf binding activity is due to a loss in initiation factor activity and not to a loss of any 40‐S subunit activity.The decreased inhibitory activity of dsRNA at high salt concentrations is due both to a delay in the formation of a dsRNA‐activated inhibitor, and to a reduced effect on protein synthesis of the inhibitor.

The effects of salt on the TATA binding protein-DNA interaction from a hyperthermophilic archaeon

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions

The adsorption of alkylpyridinium surfactants on Aerosil OX50 silica has been studied as a function of surfactant concentration, aliphatic chain length, pH, and ionic strength in solution. The adsorption isotherms are complemented by electrophoretic mobility measurements. The experimental results have been compared with calculations based on the SCFA theory for adsorption on a charge-regulating surface. The experimental results and SCFA calculations both show that the isotherms of the cationic surfactant on silica, obtained at different salt concentrations, have a common intersection point (cip). As was shown before by surface charge adaptation experiments and is now confirmed by the theoretical calculations, aliphatic segments have a moderate affinity to the surface due to hydrophobic interactions. These interactions result in a flat orientation of the surfactant molecules in the adsorbed layer. The shape of the adsorption isotherms depends on the salt concentration. The first pseudoplateau observed at low salt concentrations vanishes at high salt concentration. At low salt concentrations, electrostatic repulsions between “head-on” adsorbed headgroups and local crowding prevent the aggregation process in the adsorbed layer and inhibit further adsorption in the region of the first pseudoplateau. At high salt concentrations, the adsorption starts later due to the screening of the surface charge by the salt ions, and it increases steeper due to the diminished lateral repulsions between surfactant molecules. The common intersection point in the isotherms measured at different salt concentrations marks the transition from a flat monolayer to a flat bilayer.

Conformation of single polyelectrolytes in tetravalent salt solutions is investigated under the framework of a coarse-grained model, using Langevin dynamics simulations. The chain size, studied by the radius of gyration, shows three different variational behaviors with salt concentration, depending on the chain stiffness. According to the size variations, polyelectrolytes of fixed chain length are classified into three categories: (1) flexible chain, for which the variation shows a curve similar to a tilted L, (2) semiflexible chain, whose curve resembles U, and (3) rigid chain, for which the curve is a straight line. The wormlike chain model with persistence length predicted by the Odijk-Skolnick-Fixman theory is found to be able to qualitatively describe the end-to-end distance at low salt concentration not only for semiflexible and rigid chains but also for flexible chain. In a low salt region, a flexible polyelectrolyte extends more significantly than a semiflexible chain, in reference of the size of their uncharged counterparts, and in a high salt region, regardless of chain stiffness, a chain attains a dimension comparable to that of its neutral polymer. The chain stiffness influences both the local and the global chain structures. A flexible chain exhibits a zigzagged local structure in the presence of salt ions, and the condensed structure is a disordered, random globule. A semiflexible chain is locally smooth, and the condensed structure is orderly packed, taking a form such as hairpin or toroid. Moreover, the chain stiffness can also affect the nature of the coil-globule transition. The transition occurred in a discrete manner for semiflexible chain, whereas it occurred in a continuous way for flexible chain. This discrete feature happened not only at low salt concentration when a semiflexible chain collapsed but also at high salt concentration when the collapsed chain is reexpanded. At the end, the effects of chain stiffness and salt concentration on the conformation of single polyelectrolytes are summarized in a schematic state diagram.

Initiation of polymerization by ammonium trichloracetate in the presence of copper derivatives — A tracer study

Summary The effect of sodium chloride concentration and pH on the kinetics of the neutralization of Cypridina luciferase by antiluciferase γ-globulin has been investigated. Determination of the rate of neutralization in the presence of excess γ-globulin showed that approximately 60% of the initial activity was neutralized according to pseudo-first order kinetics. The rate of neutralization increased on lowering the sodium chloride concentration and decreased on increasing the salt concentration. The rate also increased in going from pH 7.0 to 5.4 at low salt concentrations, but was insensitive to variation in hydrogen ion concentration at high salt concentrations. At low ionic strengths, the observed results conformed to the predictions of the Brönsted theory of the primary salt effect on reaction kinetics. Consequently, the rate changes observed in the presence of sodium and chloride ions were considered as being due to their effect on the “activity” of an intermediate “activated complex.” Alternatively, the effect of salt and hydrogen ion concentration on reaction rate was also considered in terms of a modified diffuse double layer affecting the collision frequency of the molecules. Determination of the rate constants at varying hydrogen ion concentrations, and subsequent analysis of the data in terms of an interaction between a single ionic species of luciferase and antibody, indicated the involvement of active groups possessing pK1 = pK2 = pK3 = 5 and pK4 ≧ 8.

Water-based electrolytes provide safe, reliable, and cost-effective energy storage solutions; however, their application in aqueous lithium-ion batteries is hindered by low energy density and short cycling life due to the limited electrochemical stability window. While high lithium salt concentrations can mitigate some of these issues, they often lead to increased solvent viscosity and higher costs, limiting commercialization. In this study, a boron-stabilized anisotropic polyvinyl alcohol (PVA) hydrogel electrolyte, referred to as BaP, is proposed to address the challenges related to high lithium salt (LiTFSI) concentrations. Due to the Hofmeister effect, the BaP water-in-polymer electrolyte can retain a high concentration of lithium salt even when low concentrations of lithium salt are used. Briefly, the BaP promotes the salting-in phenomenon of Li ions, while the TFSI ions induce salting-out, allowing BaP to synergistically achieve high lithium salt concentrations. Due to these unique characteristics, the BaP hydrogel exhibits a wide electrochemical stability window similar to that of highly concentrated electrolytes, enabling stable operation in a LiMn2O4||Li4Ti5O12 full cell by suppressing hydrogen evolution. Moreover, the biodegradability of BaP contributes to the development of a more environmentally friendly battery system.

Red halophilic Archaea (Halobacteriaceae) are found worldwide in environments in which salt concentrations exceed 2.5-3 M, such as salt lakes and solar saltern crystallizer ponds. They also occur on products such as salted fish and salted hides. In salt lakes they are often found in numbers sufficiently high to impart a reddish color, due to their content of carotenoid and possibly also retinal pigments. KCl is accumulated intracellularly in molar concentrations to provide osmotic balance with the salinity of the outside medium. The presence of high intracellular salt concentrations requires special adaptations of the cells’ enzymes and other macromolecules to be stable and active under physiological conditions. One of these adaptations is the presence of a large excess of acidic amino acids in the proteins, and a paucity of hydrophobic residues. In the absence of salt these proteins are unstable and unfold, thus the constant presence of high salt concentrations is required for the cells’ integrity and viability. Within the limitations dictated by the requirement for a minimal and high salt concentration, the physiological diversity among the members of the Halobacteriaceae is substantial. In addition to respiratory metabolism on a variety of carbon and energy sources, species may live by anaerobic respiration using different electron acceptors, some are able to ferment arginine, and certain species may also use light energy via the light driven primary ion pumps bacteriorhodopsin and halorhodopsin. Some species are adapted to life in the extremely high magnesium and calcium concentrations of the Dead Sea. Analysis of 16S rRNA nucleotide sequences, combined with renewed attempts in recent years to isolate additional types, has yielded a complex picture of phylogenetic relationships within the group. In spite of the progress made, those species most abundant in such environments as saltern crystallizer ponds still defy our attempts to isolate them, as shown by the fact that direct amplification of halophilic archaeal 16S rRNA sequences from the brines yields phylotypes different from the cultured species of halophilic Archaea.

Effects of Buffer Loading for Electrospray Ionization Mass Spectrometry of a Noncovalent Protein Complex that Requires High Concentrations of Essential Salts

Protein adsorption on novel acrylamido-based polymeric ion-exchangers: III. Salt concentration effects and elution behavior

In this work a confocal Raman microspectrometer is used to investigate the influence of Na(+) and Mg(2+) ions on the DNA structural changes induced by low pH. Measurements are carried out on calf thymus DNA at neutral pH (7) and pH 3 in the presence of low and high concentrations of Na(+) and Mg(2+) ions, respectively. It is found that low concentrations of Na(+) ions do not protect DNA against binding of H(+). High concentrations of monovalent ions can prevent protonation of the DNA double helix. Our Raman spectra show that low concentrations of Mg(2+) ions partly protect DNA against protonation of cytosine (line at 1262 cm(-1)) but do not protect adenine and guanine N(7) against binding of H(+) (characteristic lines at 1304 and 1488 cm(-1), respectively). High concentrations of Mg(2+) can prevent protonation of cytosine and protonation of adenine (disruption of AT pairs). By analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high magnesium salt protect the N(7) of guanine against protonation. A high salt concentration can prevent protonation of guanine, cytosine, and adenine in DNA. Higher salt concentrations cause less DNA protonation than lower salt concentrations. Magnesium ions are found to be more effective in protecting DNA against binding of H(+) as compared with calcium ions presented in a previous study. Divalent metal cations (Mg(2+), Ca(2+)) are more effective in protecting DNA against protonation than monovalent ions (Na(+)).