Side-chain effect on conformation of ionizable polypeptides in aqueous solution

In order to study the effect of side-chain length on the conformation of polypeptides, conformational changes of various ionic polypeptides with various lengths of side chain, poly-N^ε-glutaryl-L -lysine (PGL), poly-N^δ-glutaryl-L -ornithine (PGO), poly-N^ε-succinyl-L -lysine (PSL), and poly-N^δ-succinyl-L -ornithine (PGO), were investigated by ORD, potentiometric titration, and dilatometric measurements in aqueous solution. The results of optical rotation and potentiometric titration measurements indicate strongly that the α-helix stability increases in the sequence PSO < PSL ～ PGO < PGL, which corresponds to increased side-chain length. The volume change associated with the helix–coil transition also increased in the above sequence. This series of polymers seems to be more hydrophobic compared with poly-L -glutamic acid or poly-L -lysine, as suggested from the values of enthalpy and entropy changes for coil–helix transitions.     

Texture formation in DNA films with alkali metal chlorides

The formation of textures in DNA films with LiCl, NaCl, KCl, RbCl, and CsCl salts has been studied. The films are prepared by evaporation of water solution with highly polymerized calf thymus DNA and excess salt of specific type. For DNA solution with 10 mM concentration of NaCl, KCl, and RbCl the films with dendritic textures have been obtained, whereas in case of CsCl the textures in the films appear only at 30 mM concentration of excess salt in the initial solution. In the solution with LiCl, the textures in DNA films have not been observed within the whole range of concentration of excess salt under consideration. The analysis of parameters of DNA films with different salts has showed that evaporation of solution leads to crystallization of salt ions on DNA macromolecule and formation of DNA‐salt complexes. Electrostatic energy of the system of crystalline ordered ions and charges of DNA chains has been estimated to study the stability of DNA‐salt complexes. The results obtained for different salts have been showed that the presence of DNA macromolecule enhances crystallization as compared with solution without DNA. The property of excess salt to form the crystalline structures has been found to decrease in the following order: KCl > NaCl > RbCl > CsCl > LiCl. The results of estimation are in good agreement with the experimentally observed dependence of texture formation on excess salt type. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 508–516, 2013.     

Activity coefficients of salts in highly concentrated protein solutions: I. Alkali chlorides in isoionic bovine serum albumin solutions

In order to understand the thermodynamic state of simple salts in living cells, the mean activity coefficients of LiCl, NaCl, KC1, RbCl, CsCl were determined in concentrated isoionic bovine serum albumin (BSA) solutions by use of the EMF method with ion exchange membrane electrodes. The protein concentration range extended up to 22 wt %, whereas the salt concentration was kept constant at 0.1 mole per kilogram water. These solutions may be regarded as crude but appropriate model systems for the cytoplasm of cells as far as type and magnitude of the macromolecular component influence on the chemical potential of the salts is concerned. The mean stoichiometric activity coefficients of the alkali chlorides in the isoionic BSA solutions decreased linearly with the protein molality; this decrease, however, did not exceed ca. 10% compared with the pure 0.1 molal salt solutions. Only very small differences in the behaviour of the different alkali chlorides were observed. The results may be interpreted by the superposition of the effects of specific Cl^? ion binding to BSA and BSA bound “non-solvent” water with probably electrostatic long range interactions of the BSA(Cl^?)_v polyions with the salt ions in solution. The resulting mean activity coefficients, corrected for ion binding and non-solvent water, showed a very slight linear dependence on the protein concentration. The departure from the value in the pure 0.1 molal salt solutions did not exceed ± 2%.     

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

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

Potentiometric titrations and the helix-coil transition of poly(L-glutamic acid) and poly-L-lysine in aqueous salt solutions

The objective have been to establish if those ions which are known to change the stability of the structure of proteins, have any influence on the properties of ionizable polypeptides. Potentiometric titrations and complementary optical rotation data are presented for aqueous solutions of poly-L -lysine (PLL) in the presence of KSCN, KCl, and KF, and for poly(L -glutamic acid) (PLGA) in the presence of KSCN, KCl, and LiCl. The following measured quantities which are affected by salt concentration were obtained: intrinsic pK (pK₀), slope of pK_app versus degree of ionization (α) curves, the degree of ionization at which the helix to coil transition occurs, and the free energy of this transition for the uncharged molecule (δG°_hel). The effects of nonspecific salts (KCl and LiCl for PLL and KSCN and KCl for PLGA) are small, and about, as expected from general electrostatic considerations. In line with the observations made with isoelectric and cat ionic collagen, specific, effects were noted with KSCN–PLL and with LiCl–PLGA. In the presence of KSCN, the poly-L -lysine helix becomes stabilized at much lower degree of ionization than in the presence of KCl, and the slope of the pK_app versus α plots is greatly reduced. However, ΔG°_hel (for the uncharged molecule) is not affected, and pK₀ is only slightly higher. We interpret these data in terms of binding of SCN^? primarily to the side-chain amino groups (both to R? NH₃⁺ and to R? NH₂) solutions. (L -glutamic acid) in LiCl solution has its transition at the same α value as in KCl solution. However, both the slopes of the pK_app versus α plots and the absolute values of ΔG°_hel are lower than in KCl solution. We interpret these results in terms of binding of Li⁺ to side chains as well as to the peptide bond.     

Effect of Salts on Growth and Pectinase Production by Halotolerant Yeast, <Emphasis Type="Italic">Debaryomyces nepalensis</Emphasis> NCYC 3413

In this study, Debaryomyces nepalensis NCYC 3413 isolated from rotten apple was studied for its halotolerance and its growth was compared with that of Saccharomyces cerevisiae in high salt medium. The specific growth rate of D. nepalensis was not affected by KCl even up to a concentration of 1 M, whereas NaCl and LiCl affected the growth of D. nepalensis. Among all tested salts, LiCl showed maximum inhibition on growth. At all conditions, halotolerance of D. nepalensis was much higher than that of S. cerevisiae. D. nepalensis showed maximum viability (80–100%) when grown in KCl, which was higher than with NaCl and LiCl. Pectinase production by D. nepalensis was noted at all high salt concentrations, namely, 2 M NaCl, 2 M KCl, and 0.5 M LiCl, and the maximum specific activity was observed when the strain was grown in 2 M NaCl.     

Distribution of saccharides and salts on amphoteric ion-exchange resin

An amphoteric ion-exchange resin hardly shrank in 550 and 300 g/L glucose and sodium chloride solutions, respectively; however, the bed packed with a cation-exchange resin shrank considerably. From the distribution coefficients of some saccharides, the swelling pressure of the amphoteric ion-exchange resin was estimated to be 2.0 MPa at 25 °C. The distribution coefficients of glucose, galactose, fructose, and mannose were independent of their concentration and were about 0.621. On the other hand, the apparent distribution coefficients of NaF, NaCl, NaBr, NaI, LiCl, KCl, and CsCl largely depended on concentration. A model for the distribution of salts on the amphoteric resin was proposed, assuming an interaction between the anion of the salt and the positively charged fixed ions with binding constant B. The B values of the chloride salts were nearly the same (1.69–2.94 L/mol), while the values of the sodium salts were largely different depending on the anion.     

Studies on the interaction between alkali metal cations and polyion by sound velocity

The sound velocities in polyelectrolyte solutions were measured at various concentrations of added salts. When aqueous solutions of tetra (n-butyl)ammonium polyacrylate were titrated with concentrated solutions of LiCl, NaCl, KCl or CsCl, the sound velocity, i.e., the adiabatic compressibility of the solution, did not change linearly with added salt concentration, but showed a breaking point. The degrees of counterion binding on polyacrylate ion estimated from the breaking points were 0.25-0.30, independent of cation species. In polystyrenesulfonate, moreover, no Na+ binding was detected from such sound velocity measurements.     

X-ray studies on the conformation of poly-N -carbobenzoxy-L- , -diaminobutyric acid and poly-N -carbobenzoxy-L-ornithine

X-ray diagrams from oriented films and fibers of poly-N^γ-carbobenzoxy-L -α,γ-diaminobutyric acid (PCLB) and of poly-N^δ-carbobenzoxy-L-ornithine (PCLO) have been examined. The conformation in the solid state for both polymers is that of an α-helix, 18/5 for PCLB and 11/3 for PCLO, respectively. Furthermore, while the PCLB molecules are packed in a trigonal lattice whose dimensions, on hexagonal axes, are a = 27.5 and c = 27.0 Å, the PCLO unit cell is monoclinic with a = 23.3, b = 22.7, c = 16.2 Å, and γ = 119.2°.     

Synthesis and conformational properties of polypeptides containing long aliphatic side chains. Poly(Nepsilon-stearyl-L-lysine) and poly(Nepsilon-pelargonyl-L-lysine).

Poly(N^ε-stearyl-L -lysine) and poly(N^ε-pelargonyl-L -lysine) were synthesized both by polymerization of N^ε-pelargonyl and N^ε-stearyl-L -lysine NCA and by acylation of poly(L-lysine) with pelargonyl and stearyl chloride. This second route has proven to be very useful, since completely acylated polymers are obtained in almost quantitative yield, whereas the usual scheme of preparation of ε protected poly(L-lysine) cannot easily be applied due to solubility problems. Poly(N^εpelargonyl and stearyl-L -lysine) are soluble in alcohols containing linear aliphatic chains such as n-butanol and n-octanol and in mixtures of these alcohols with hydrocarbons such as n-hexane and n-heptane. Both polymers show an α-helical conformation in the above solvents, which can be disrupted upon addition of sulfuric acid. Also in the solid state, poly(N^ε-stearyl-L -lysine) and poly(N^ε-pelargonyl-L -lysine) show X-ray diffraction patterns typical of order structure.     

Influence of Electrolytes on the Thicknesses of the Phospholipid Bilayers of Lamellar Lecithin Mesophases

Over a wide range of water contents, aqueous lecithin-water mixtures are mesophases in which lecithin bilayers alternate with water layers. This paper reports on low-angle X-ray diffraction measurements of the effects of electrolytes, at 1.0 N concentration, on the thicknesses of the bilayers in mesophases formed by the synthetic lecithin: 1-octadec-9-enyl-2-hexadecylglycerophosphocholine. With solutions of LiCl, NaCl, Na₂SO₄, KCl, and CsCl, the bilayer thicknesses are less than with pure water. The maximum reduction in bilayer thickness with these electrolytes is about 10% and occurs with mesophases of high content of KCl and CsCl solutions. With HCl solutions the bilayer thicknesses are about 5% greater than with pure water, and with CaCl₂ solutions the bilayer thicknesses are about the same as with pure water. The maximum amount of solution which can be mixed with lecithin before a second, purely aqueous phase is formed is also affected by electrolytes, the order for the various 1.0 N solutions being CsCl = KCl > NaCl > Na₂SO₄ > (pure water) = LiCl > CaCl₂.     

MICRURGICAL STUDIES IN CELL PHYSIOLOGY : V. THE ANTAGONISM OF CATIONS IN THEIR ACTIONS ON THE PROTOPLASM OF AM?BA DUBIA.

I. The Plasmalemma. 1. On the plasmalemma of amebæ CaCl₂ antagonizes the toxic action of LiCl better than it does NaCl, and still better than it does KCl. MgCl₂ antagonizes the toxic action of NaCl better than it does LiCl and still better than it does KCl. 2. CaCl₂ antagonizes the toxic action of LiCl and of KCl better than does MgCl₂: MgCl₂ antagonizes NaCl better than does CaCl₂. II. The Internal Protoplasm. 3. The antagonizing efficiency of CaCl₂ and of MgCl₂ are highest against the toxic action of KCl on the internal protoplasm, less against that of NaCl, and least against that of LiCl. 4. CaCl₂ antagonizes the toxic action of LiCl better than does MgCl₂: MgCl₂ antagonizes the toxic action of NaCl and of KCl better than does CaCl₂. 5. LiCl antagonizes the toxic action of MgCl₂ on the internal protoplasm more effectively than do NaCl or KCl, which have an equal antagonizing effect on the MgCl₂ action. III. The Nature of Antagonism. 6. When the concentration of an antagonizing salt is increased to a toxic value, it acts synergistically with a toxic salt. 7. No case was found in which a potentially antagonistic salt abolishes the toxic action of a salt unless it is present at the site (surface or interior) of toxic action. 8. Antagonistic actions of the salts used in these experiments are of differing effectiveness on the internal protoplasm and on the surface membrane.     

Effects of alkali ions on myosin conformation

We have used two probes to study the effects of alkali ions on the conformation of myosin. One was paramagnetic, the “spin label” N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide, which binds primarily to SH groups; and the other was fluorescent, l-anilino-8-naphthalenesulfonate, which binds to an apolar niche. The bonding of the spin label to myosin was carried out in 0.6M LiCl, 0.6M NaCl, or 0.6M KCl, and the resulting labeled myosin was studied in the same medium in which the myosin was labeled as well as in other alkali chlorides. The electron paramagnetic resonance spectra of the spin label showed that the structure of myosin in the vicinity of the labeled groups differed in the various salts. The protein surface in the region of the labeled groups restricted the rotational freedom of the spin label more in KCl than in any of the other salts. Although ions are known to influence the properties of myosin, our results show that these ions also effect the molecular structure. The fluorescence of l-anilino-8-naphthalenesulfonate, noncovalently attached to myosin in the presence of alkali chlorides, decreased progressively with increasing size of the cations, again showing the protein structure near the probe attachment to be a function of the cation, in the solvent. Ca²⁺ quenched the fluorescence of the bound probe, indicating an interaction between Ca²⁺ and the myosin molecule. The effect of Ca²⁺ on the fluorescence was greatest in KCl.     

Osmotic Effects on Membrane Permeability in a Marine Bacterium

When cells of Alteromonas haloplanktis 214 (ATCC 19855) were preloaded with α-[¹⁴C]aminoisobutyric acid or the K⁺ in the cells was labeled with ⁴²K by incubation in a buffered salt solution containing 0.05 M MgSO₄, 0.01 M KCl, and 0.3 M NaCl, the cells retained their radioactivity when resuspended in the same salt solution. When NaCl was omitted from the solution, 80 to 90% of the radioactivity was lost from the cells. Cells suspended at intermediate concentrations of NaCl also lost radioactivity. New steady-state levels of the intracellular solutes were established within 15 s of suspending the cells; the percentage of radioactivity retained at each level decreased proportionately as the osmolality of the NaCl in the suspending solution decreased. With minor variations in effectiveness, MgCl₂, LiCl, and sucrose could substitute for NaCl on an equiosmolal basis for the retention of radioactivity by the cells. KCl, RbCl, and CsCl were appreciably less effective as replacements for NaCl, particularly when their osmolalities in the suspending solutions were low. The amount of α-[¹⁴C]aminoisobutyric acid taken up by the cells at the steady-state level increased to a maximum as the NaCl concentration in the suspending medium increased to 0.3 M. At suboptimal levels of NaCl, either LiCl or sucrose could substitute for NaCl in increasing the steady-state levels. The results obtained indicate that the porosity of the cytoplasmic membrane of this organism is determined by the difference between the osmotic pressure of the cytoplasm and the suspending medium. The lesser effectiveness of K⁺, Rb⁺, and Cs⁺ than Na⁺, Li, or Mg²⁺ in permitting the retention of solutes by the cells is attributed to the greater penetrability of the hydrated ions of the former group through the dilated pores of a stretched cytoplasmic membrane.     

Salt-tolerance in plants. I. Ions, compatible organic solutes and the stability of plant ribosomes

Abstract Polysomes and ribosomes recovered from a number of plant species were tested for stability when incubated at 25°C in salt solutions in the absence of ATP and initiation factors. Stability was assessed by sucrose density gradient analysis. The stability was inversely proportional to salt concentrations above 125 mol m⁻³ KCl. Polysomes were less stable in the presence of Na⁺ than K⁺ salts, and were much less stable in Cl⁻ than in acetate salts. Polysomes from Triticum aestivum. Hordeum vulgare, Capsicum annuum, Helianthus annuus. Pisum sativum, Atriplex nummularia, Beta vulgaris, Cladophora sp., Enteromorpha sp. and Corallina cuvieri were similarly sensitive to KCl. Polysomes from Ulva lactuca were more sensitive than the other species. Cytoplasmic and plastid polysomes from T. aestivum were similarly unstable in 500 mol m⁻³ KCl. Unprogrammed ribosomal subunit couples from T. aestivum, B. vulgaris and U. lactuca showed Mg²⁺-dependent conformational instability and dissociation in KCl. Slight differences in ribosomal stability were observed between species, but these were unrelated to the salt tolerances of the plants. The ‘compatible’ organic solutes, glycinebetaine and proline, failed to reduce ion-induced instability. Ribosome yield and polysome profiles were similar in leaves of B. vulgaris containing significantly different levels of both Na⁺ and Cl⁻ after growth in media containing 50 or 200 mol m⁻³ NaCl. The results are consistent with the hypothesis that plants maintain a cytoplasmic solute environment that is compatible with ribosomal stability.     

Effect of agents affecting water structure on the uptake of saccharides by agar and polyacrylamide gels

Uptake of D -xylose, D -glucose, lactose, and dextran 10 was studied in 3% and 12.5% polyacrylamide gels in the presence of 2 M LiCl, 2 M NaCl, 2 M KCl, 2 M RbCl, 2 M CsCl, 2 M MgCl₂, 2 M CaCl₂, 1 M NaF, 1 M NaI, 0.5 M guanidine thiocyanate, 0.5 M guanidine sulphate, and 0.5 M N,N′-diethylurea. With the exception of N,N′-diethylurea and, in some cases, LiCl, which had an accelerating effect, the compounds retarded considerably the uptake of saccharides by agar gel but had only a slight effect in polyacrylamide. The nature of the gel is thus of primary importance in interactions of this type. According to the magnitude of their effect on saccharide uptake, the salts used were arranged into several series. An attempt was made to correlate the effects of individual salts with their hydration properties. The strongest effect was found to be exerted by KCl, RbCl, and CsCl, i.e., by chlorides of cations possessing negative hydration shells.     

The effects of aqueous neutral-salt solutions on the melting temperatures of deoxyribonucleic acids

The helical stability of a variety of DNA samples, ranging in base composition from 0 to 72 mole-% GC, has been studied by heat denaturation at neutral pH in increasing concentrations of LiCl, NaCl, KCl, CsCl, Li₂SO₄, and K₂SO₄. The variation of melting temperature with average base composition, dT_m/dX_GC, was found to decrease drastically in the concentrated salt media, e.g., from 41°C in 0.006M LiCl to 29°C in 3.2M LiCl, and from 39°C in 0.003M Li₂SO₄ to 18°C in 1.6M Li₂SO₄. At the same time, the thermal transition is much more cooperative in the concentrated salt solutions than at low ionic strength. Indeed, at limiting salt concentrations, the transition breadth seems to reach a minimum value irrespective of the compositional heterogeneity of the DNA samples. Attempts to correlate the observed decrease of dT_m/dX_GC with predicted changes in the enthalpy of melting, deduced from a simple theoretical treatment, experimental data on the binding of counterions and water to DNA, and experimental data on thermal denaturation, were unsuccessful. However, the strongly reduced composition dependence of the melting temperature can be understood in terms of a destabilizing effect of the concentrated salt media on GC-base pairs. It is suggested, though not proven, that the destabilization involves the displacement of water molecules from the DNA helix.     

Salt-induced conformational change of random copolymers (Lys50Tyr50)n and (Lys50Phe50)n studied by 1H- and 13C-nuclear magnetic resonances. Aggregation behavior of helical segments

¹H- and ¹³C-nmr studies of conformational transitions of random amino acid copolymers containing aromatic residues (Lys⁵⁰Tyr⁵⁰)_n and (Lys⁵⁰Phe⁵⁰)_n in the presence of neutral salts were performed to serve as models of the aggregation behavior of polypeptides of biological significance. The ¹H and ¹³C signal intensities of Tyr and Phe residues decreased preferentially with increasing concentration of neutral salts such as NaCl and NaClO₄. This behavior contrasts with that of (Lys)_n in the presence of similar neutral salts, where the displacement of the ¹³C signal is clearly seen on transition from the random-coil to the helical conformation. On the basis of the previous conformational studies, the loss of the peak areas is ascribed to the presence of immobilized helical segments by hydrophobic interaction between aromatic side chains. The remaining resonances are due to the residual random-coil regions, since the values of nuclear Overhauser enhancements and chemical shifts are unchanged in the presence and absence of the neutral salts.     

Toluidine blue binding capacity of heterochromatin and euchromatin of Triatoma injestans Klug

Summary Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO₃, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

Structure of phenylalanine-accepting transfer ribonucleic acid and of its environment in aqueous solvents with different salts

Yeast tRNA(Phe) was studied in different salt-containing solvents by UV absorbance and small-angle neutron scattering (SANS). This extends results obtained previously in NaCl and KCl solutions [Li, Z.-Q., Giegé, R., Jacrot, B., Oberthür, R., Thierry, J. C., & Zaccai, G. (1983) Biochemistry 22, 4380-4388]. As expected, at low concentrations of all salts studied, the tRNA molecule is unfolded. The importance of specific counterion interactions and the flexibility of the macromolecule are emphasized by the observation that it cannot take up its folded structure in N(CH3)4Cl solvents, even when that salt concentration is increased to 1 M, in the absence of Mg ions. In CsCl solvents, on the other hand, the folded conformation is obtained in salt concentrations above about 0.2 M, similar to NaCl or KCl. By a comparison of SANS results in CsCl H2O and CsCl 2H2O solvents with the data from NaCl and KCl solvents, thermodynamic and structural parameters were derived for the solvated macromolecule. All the data are accounted for, quantitatively, by a model for the particle in NaCl, KCl, or CsCl solution made up of tRNA76-, closely associated with 76 positive hydrated counterions, surrounded by an aqueous solvent layer that excludes salt (and, therefore, of density different from that of bulk solvent). The mass of water in that layer depends on salt concentration, and the values found are consistent with those predicted by the Donnan effect.