Kinesin undergoes a 9 S to 6 S conformational transition.

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.     

Effect of the medium on functional and structural properties of serum albumins. IV. The state of human serum albumin in the pH range from 5 to 10

In order to investigate the effects of temperature and ionic strength on the N-B-transition and the alkaline denaturation of the human serum albumin, the pH-dependences of fluorescence position and relative yield of Trp-24 and of protein bound dye ANS were measured. The measurements were carried out at temperatures from 10 to 45 degrees C and ionic strengths (NaCl) from 0.001 to 0.2. The pH-induced structural transitions have different realization in environments of tryptophanyl and tightly bound ANS. The alkaline denaturation does not change the Trp-214 fluorescence. The N-B-transition gives rise to the slight polarity and/or mobility lowering in the Trp-214 environment (the shorter-wave-length spectral shift). Increase in the temperature and ionic strength induces the shift of the transition midpoint from ca. 8 to 8.7 and reduces the spectral shift amplitude. At low ionic strengths, the new structural transition in the Trp-214 environment is observed at pH change from 6.7 to 5.7. This transition is not observable using ANS fluorescence. The N-B-transition is accompanied by an enhancement and longer-wavelength shift of the ANS fluorescence spectra. The transition midpoint is independent of temperature, but is shifted to lower pH values at a decrease of ionic strength value. At ionic strengths less than or equal to 0.01 the shorter-wavelength spectral shift is seen at pH from 7.5 to 9, which seems to reflect the disulfide B-A-isomerisation. The alkaline denaturation gives rise to the sharp quenching of ANS fluorescence, probably due to the ANS binding site decomposition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversibility of the low-salt transition of chromatin core particles.

The low-salt transition of chromatin core particles is reversible if the monovalent cation concentration is kept above 0.2 mM. Exposure of the particles to salt concentrations below this value results in a nonreversible secondary transition. The nonreversible changes are relatively slow with a half-time of about 15 minutes. Once exposed to such low ionic strength, the particles then begin to refold with increasing salt in at least two steps over a much higher ionic strength range than is required for the usual low-salt transition. The refolding is very fast, with a half-time less than a minute. Small differences between particles which had or had not been exposed to very low salt persist even when the particles are returned to near physiological ionic strengths.     

Cooperative lipid-protein interaction. Effect of pH and ionic strength on polymyxin binding to phosphatidic acid membranes.

The binding of polymyxin-B to charged dipalmitoyl phosphatidic acid membranes has been studied as function of the external pH and of the ionic strength of the buffer solution. The phase transition curves were obtained by measuring the fluorescence depolarization of diphenyl hexatriene incorporated into the membrane with temperature. The molecular process of polymyxin binding was elucidated: 1. At an ionic strength of I greater than or equal to 0.1 mol/l a three step phase transition curve is found. A high-temperature step corresponds to the non-bound lipid. A lowered phase transition concerns to protein-bound lipid domains. This again is splitted into two steps. An inner core of the domain is characterized by a lipid-protein complex which is stabilized through hydrophobic and electrostatic interactions between polymyxin and the charged lipid. This core is surrounded by an outer belt of only hydrophobically bound molecules. This part shows a lower phase transition temperature than the inner core. 2. The binding curves of polymyxin to phosphatidic acid membranes depend strongly on the ionic strength of the water phase. The cooperativity of the binding process increases with increasing ionic strength and reaches a constant value at I greater than 0.2 mol/l. The maximum fraction of bound lipid decreases with increasing ionic strength. 3. The pH of the water phase strongly influences the cooperative binding process. At pH 6 a loss of cooperativity is observed at low ionic strength. Increasing the ion concentration to I = 0.3 mol/l recuperates the cooperativity of the binding process. At pH 3.0 no cooperative binding is obtained even at high ionic strength.     

A dynamical model for phase transitions of lipids induced by calcium ions

Lipid membranes exist essentially in two different phases. A phase transition can be triggered off either by changing the temperature or, isothermally, by varying an external factor such as ionic concentration, pH, organic solvents, etc. Since the isothermal transition may be induced at physiological temperature, it may play an important regulatory role in diverse cellular functions. Based on the Landau-Ginsburg theory, the thermotropic transitions of lipids has been described by a number of models. In the present work a dynamical model for an isothermal phase transition of phospholipids induced by ionic binding is proposed. The properties of the model show that by ionic binding, phospholipids may form spatial heterogeneous distributions of lipids in fluid and crystalline phases. This heterogeneity possibly being the cause of membrane instabilities, which favour enhanced vesicle fusions observed in the presence of Ca2+.     

The temperature and pH dependence of conformational transitions of the chromatin subunit.

Hydrodynamic, spectroscopic, and chemical crosslinking studies on monomer chromatin subnits are reported as a function of ionic strength, pH, and temperature. In earlier studies, two salt-dependent conformational transitions were described (Gordon et al., Proceedings of the National Academy of Science, 75, 660, 1978). Transition one occurred between 0.7 and 2.0 mM ionic strength and transition two occurred between 5.0 and 11.0 mM ionic strength. Crosslinking at 11 mM ionic strength with formaldehyde suppressed both transitions. In this communication we report that the second transition was characterized by changes in the circular dichroism spectra in the 260--320 nm region as well as by changes in the hydrodynamic properties. As the ionic strength was increased from 5.0 to 11.0 mM, [theta]282 decreased from 2000 TO 1500 DEG CM2/DMOLE AND [THETA]295 decreased from 0 to -400 deg cm2/dmole. Both transitions occurred in the pH range from pH 6.0 to 9.2. At pH 5.0, the two ionic strength-dependent transitions were no longer observed and the characteristic changes in the circular dichroism spectra were suppressed. The spectra of the monomer subunits at pH 5.0 showed only small changes with ionic strength and resembled the spectra of the subunits at 11 mM ionic strength above pH 6.0. In order to characterize the transitions in thermodynamic terms an ionic strength near the midpoint of each transition was selected. Then, changes in s20,w and D20,w were measured as a function of temperature. These data allow an estimation to be made of the enthalpies and entropies of the transitions.     

Refolding transition of alpha-chymotrypsin: pH and salt dependence.

It is well known that alpha-chymotrypsin can exist in two major conformational states, only one of which is active. We have examined the pH (pH 2.0--11.0) and salt (ionic strength 0.01--1.0) dependence of the transition between the active and inactive forms in detail. At low pH (pH 2.0--6.0) the equilibrium is very dependent on salt concentration, with high salt concentrations effectively stabilizing the active conformation. This apparent stabilization is an artifact due to the salt-dependent dimerization of alpha-chymotrypsin, and our data show that only active species form dimers and higher aggregates. At neutral pH (6.0--8.0) dimerization is absent, yet an ionic strength dependence remains. The effects show no lyotropic order and appear to be due to preferential salt binding to the active conformation at one or possibly a few sites. Above pH 6 (pH 6.0--11.0), the pH dependence can be described by a two-ionization mechanism at all ionic strengths. We report values for all seven equilibrium constants in the proposed mechanism at four ionic strengths (mu = 0.01, 0.05, 0.2, and 1.0). The transition is the first "refolding" transition to be studied at high precision, but, even so, certain decisions about the mechanism must await higher experimental precision not available with present methods.     

Salt and temperature-dependent conformation changes in spectrin from human erythrocyte membranes.

ORD and CD measurements of spectrin, in both the dimer and tetramer association state, indicate a high proportion of alpha-helix in this protein. At temperatures below 27 degrees C and in 0.1 M NaCl, the tetramer has an apparent helix content of 73% and the dimer, 68%. The conformation of both states is dependent on salt concentration and temperature. Low ionic strength solutions of spectrin display lowered sedimentation coefficients and a decreased apparent helix content, indicating perhaps a slight refolding and expansion of the molecule. In addition, spectrin in low ionic strength solutions undergoes a broad temperature-dependent transition spread from 20 to 50 degrees C, while in the presence of salt the transition is sharp and centered on 49 degrees C. The temperature-dependent changes in low ionic strength solutions appear to parallel the dissociation of tetramer to dimer.     

Neutron scattering studies of nucleosome structure at low ionic strength

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.     

Charge-induced pretransition in phosphatidylethanolamine multilayers. The occurrence of ripple structures

The influence of pli and ionic strength on the phase transition behaviour of 1,2-dihexadecylphosphatidylethanolamine was studied calorimetrically. In the range of ionic strength from 0.75 to 1.5 M NaC1 at $\text{pH}\text{? 13}$, where the amino group of the phosphatidylethanolarnine is in the deprotonated state, resulting in one negative charge per lipid molecule, the calorimetric scan shows a pretransition before the main transition. Accompanying freeze-fracture electron microscopic studies on these preparations in the temperature range between the pre- and main transitions show a regular surface, the so-called ripple structure. These are comparable with the structures seen in phosphatidylcholine-water systems af temperatures between the pre- and main transition.     

Conformational stability of ferrocytochrome c. Electrostatic aspects of the oxidation by tris(1,10-phenanthroline)cobalt(III) at low ionic strength

At ionic strengths below 0.1 M the oxidation of horse ferrocytochrome c by tris(1,10-phenanthroline)cobalt (III) and tris(2,2'-bipyridine)cobalt(III) proceeds by a pathway which is independent of the transition metal complex concentration. Formation of an activated form of the protein appears to be rate limiting. The rate of oxidation decreases as the ionic strength increases. This dependence of the reaction rate on inert electrolyte concentration indicates that electrostatic association of anions under physiological ionic strength confers stability to the protein. The activated form of the protein, which reacts at least 10(4) times as fast as the predominant form, is thought to be a conformation of the reduced protein with an open heme crevice. Binding of the open form of ferrocytochrome c with the redox-inactive cationic transition metal complexes hexamminecobalt(III) and tris(1,10-phenanthroline)chromium(III) inhibits the oxidation by tris(1,10-phenanthroline)cobalt(III). Reactions of tris(1,10-phenanthroline)cobalt(III) with 4-carboxy-2,5-dinitrophenyllysine 13 and 72 ferrocytochromes c show no dependence on ionic strength. NMR studies at pH 7 demonstrate that ferricytochrome c is partly (15%) in the open conformation at low ionic strength. Furthermore, the interaction of redox-inert tris (1,10-phenanthroline)chromium(III) with ferricytochrome c under conditions identical to those of the kinetic studies demonstrates that the transition metal complex binds only to the open form of the protein. Titration with increasing amounts of tris(1,10-phenanthroline) chromium(III) shows changes in the NMR spectrum that are inconsistent with a single binding site.     

Ca2+ ions induce successive capacity and ion currents in BLM from phosphatidic acid as a result of phase transition

Time dependence of Ca2+-induced electric current in BLM formed from DPPA was studied at constant temperature and pH. The phase transition in BLM is accompanied by capacity current and following appearance of single ionic channels. It was shown that transferred charge was 5 nC/F, conductivity of single ionic channels--500-100 pSm.     

DNA binding to mica correlates with cationic radius: assay by atomic force microscopy.

In buffers containing selected transition metal salts, DNA binds to mica tightly enough to be directly imaged in the buffer in the atomic force microscope (AFM, also known as scanning force microscope). The binding of DNA to mica, as measured by AFM-imaging, is correlated with the radius of the transition metal cation. The transition metal cations that effectively bind DNA to mica are Ni(II), Co(II), and Zn(II), which have ionic radii from 0.69 to 0.74 A. In Mn(II), ionic radius 0.82 A, DNA binds weakly to mica. In Cd(II) and Hg(II), respective ionic radii of 0.97 and 1.1 A, DNA does not bind to mica well enough to be imaged with the AFM. These results may to relate to how large a cation can fit into the cavities above the recessed hydroxyl groups in the mica lattice, although hypotheses based on hydrated ionic radii cannot be ruled out. The dependence of DNA binding on the concentrations of the cations Ni(II), Co(II), or Zn(II) shows maximal DNA binding at approximately 1-mM cation. Mg(II) does not bind DNA tightly enough to mica for AFM imaging. Mg(II) is a Group 2 cation with an ionic radius similar to that of Ni(II). Ni(II), Co(II), and Zn(II) have anomalously high enthalpies of hydration that may relate to their ability to bind DNA to mica. This AFM assay for DNA binding to mica has potential applications for assaying the binding of other polymers to mica and other flat surfaces.     

Influence of isopropanol-water solvent mixtures on the conformation of poly-L-lysine

The helix–coil transition of poly-L -lysine (PLL) in water–isopropanol solvent mixtures has been investigated at room temperature by circular dichroism measurements. Within the range of 70%–80% isopropanol concentration (by volume), the polymer undergoes a sharp transition, characterized by the formation of a fully charged α-helical structure. On the basis of some experimental evidence the role of the organic component in solution appears more complicated than that of strengthening the intramolecular hydrogen bonds in the polymer. By analogy with the distribution of the components of alcohol–water mixtures in simple ionic systems, it is thought that only an high co-solvent concentration brings about an extensive and possible cooperative depletion of the clusters of firmly-bound water molecules in the domain of the polylelectrolyte, favoring the transition to the α-helical structure. On the other hand, CD spectral patterns show that the addition of NaCl in the alcohol-rich–water mixtures of charged poly-L -lysine gives rise to a transition from the α-helical to a β-structures conversion obeys a first-order rate law at all times, with a rate constant dependent on solvent composition and ionic strength. In these conditions, the rate of the process is close to that found for the thermally induced α–β transition. Higher polymer concentration and/or ionic strength cause a phase separation of β-PLL, suggesting that in this case interchain reactions (where hydrogen bonding should play the major role) predominate. Titration experiments on charged α-helical poly-L -lysine in 85% or 90% isopropanol mixtures confirm the occurrence of a conformational transition, which takes place within a degree of dissociation α of 0.2–0.75. The transition is accompanied by a visible turbidity, which increases as the titration proceeds. Implications of the solvent distribution around the macroion on the observed conformational phenomena are also discussed.     

Ionic switch controls the DNA state in phage λ

We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.     

Initiation of anion-selective ionic channels in bilayer membranes at lipid phase transitions

A study was carried out to electric parameters of single ionic channels initiated at phase transition of bromidmetilate 1,2-distearoyl-rac-glycero-3-(O-beta-dimethylaminoethyl)-methylphosphonate, whose molecules under conditions given below are possibly charged. It has been shown that changes of transmembrane current appear at phase transition temperature. Comparison between ionic selectivity of channels initiated at Tph.t in the membranes of DSL and its phosphate analog suggests that the channel walls initiated at phospholipid phase transitions are covered with polar groups of molecules.     

Applicability of the potentiometric titration method to the analysis of the expansion process of bovine plasma albumin in acidic solutions

To study the expansion process of bovine plasma albumin in acidic solutions, observed potentiometric titration curves at three different ionic strengths were compared with theoretical curves, using the radii of the protein determined by small angle X-ray scattering (SAXS). From the comparison, it was concluded that the expansion is completed via two different transitions and that the conformation of the protein before the first transition is stable and common at all ionic strengths, whereas the form of the protein becomes a more swollen and unstable one after the first transition. Moreover, the chargeindependent part of the standard free energy change, ΔG°, in the first transition was estimated from the potentiometric titration curves. The numerical value of ΔG° is 2350 ± 50 cal/mol., which is very small compared with the corresponding one for ordinary biopolymers.     

Synthesis of room temperature ionic liquids from carboxymethylated chitosan

Natural oligosaccharide-derived room temperature ionic liquids (RTILs) were prepared from 1-ethyl-3-methylimidazolium hydroxide (EMIM·OH) and carboxymethylated chitosan (CM-chitosan) by acid–base neutralization reaction. These EMIM·CM-chitosan ionic liquids exhibited good ionic conductivity and thermal stability, as well as low glass transition temperature, implying their potential wide applications in direct electrochemistry, biosensors, and biocatalysis.     

Equilibrium and Kinetics of DNA Overstretching Modeled with a Quartic Energy Landscape

It is well known that the dsDNA molecule undergoes a phase transition from B-DNA into an overstretched state at high forces. For some time, the structure of the overstretched state remained unknown and highly debated, but recent advances in experimental techniques have presented evidence of more than one possible phase (or even a mixed phase) depending on ionic conditions, temperature, and basepair sequence. Here, we present a theoretical model to study the overstretching transition with the possibility that the overstretched state is a mixture of two phases: a structure with portions of inner strand separation (melted or M-DNA), and an extended phase that retains the basepair structure (S-DNA). We model the double-stranded DNA as a chain composed of n segments of length l, where the transition is studied by means of a Landau quartic potential with statistical fluctuations. The length l is a measure of cooperativity of the transition and is key to characterizing the overstretched phase. By analyzing the different values of l corresponding to a wide spectrum of experiments, we find that for a range of temperatures and ionic conditions, the overstretched form is likely to be a mix of M-DNA and S-DNA. For a transition close to a pure S-DNA state, where the change in extension is close to 1.7 times the original B-DNA length, we find l ≈ 25 basepairs regardless of temperature and ionic concentration. Our model is fully analytical, yet it accurately reproduces the force-extension curves, as well as the transient kinetic behavior, seen in DNA overstretching experiments.     

Equilibrium and Kinetics of DNA Overstretching Modeled with a Quartic Energy Landscape

It is well known that the dsDNA molecule undergoes a phase transition from B-DNA into an overstretched state at high forces. For some time, the structure of the overstretched state remained unknown and highly debated, but recent advances in experimental techniques have presented evidence of more than one possible phase (or even a mixed phase) depending on ionic conditions, temperature, and basepair sequence. Here, we present a theoretical model to study the overstretching transition with the possibility that the overstretched state is a mixture of two phases: a structure with portions of inner strand separation (melted or M-DNA), and an extended phase that retains the basepair structure (S-DNA). We model the double-stranded DNA as a chain composed of n segments of length l, where the transition is studied by means of a Landau quartic potential with statistical fluctuations. The length l is a measure of cooperativity of the transition and is key to characterizing the overstretched phase. By analyzing the different values of l corresponding to a wide spectrum of experiments, we find that for a range of temperatures and ionic conditions, the overstretched form is likely to be a mix of M-DNA and S-DNA. For a transition close to a pure S-DNA state, where the change in extension is close to 1.7 times the original B-DNA length, we find l ≈ 25 basepairs regardless of temperature and ionic concentration. Our model is fully analytical, yet it accurately reproduces the force-extension curves, as well as the transient kinetic behavior, seen in DNA overstretching experiments.